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Effect of aclarubicin on contractile response of rat aorta
European Journal of Pharmacology, 167 (1989) 177-180 Elsevier
177
EJP 20442 Short communication
Effect of aclarubicin on contractile response of rat aorta I c h i r o W a k a b a y a s h i *, K a t s u h i k o H a t a k e a a n d E i z o K a k i s h i t a Second Department of lnternal Medicine, and 1 Department of Legal Medicine, Hyogo Collegeof Medicine, Hyogo, 663, Japan
Received 6 June 1989, accepted 20 June 1989
The effect of aclarubicin on vasocontractility was investigated using aortic strips isolated from rats. The aortic strips from rats injected i.p. with aclarubicin (4 mg/kg body weight per day for 5 consecutive days) showed diminished contractile responses to KC1 and phenylephrine in comparison with the controls injected with 0.9% saline. In vitro preincubation of rat aorta with aclarubicin (20-80 #g/ml) attenuated the aortic contractile responses to KC1 and phenylephrine compared with the control preincubated with 0.9% saline. These results suggest that aclarubicin reduces the contractility of vascular smooth muscle directly. Aclarubicin; Anthracycline antibiotics; Vasoconstriction
1. Introduction Anthracycline derivatives have a potent antineoplastic action, however their cardiotoxicity limits their utility for the treatment of neoplastic diseases (Kantrowitz and Bristow, 1984). Aclarubicin is an anthracycline derivative isolated from Streptomyces galilaeus (Old et al., 1975). It was reported that morphological changes in the myocardium of animals treated with aclarubicin are less pronounced than those in doxorubicintreated animals (Wakabayashi et al., 1980). There are many reports on the cardiac effect of anthracycline drugs, but only few reports on their vascular effect. We investigated the effect of aclarubicin on vasocontractility in ex vivo and in vitro experiments using the aortic strips isolated from rats.
* To whom all correspondence should be addressed: Second Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho,Nishinomiya, Hyogo, 663, Japan.
2. Materials and methods 2.1. E x vivo study
Male Wistar rats (400-450 g) received an i.p. injection of 4 m g / k g body weight per day of aclarubicin diluted with 0.9% sterile saline for 5 consecutive days. Control rats received Lp. the same volume of 0.9% sterile saline. On the 6th day after the start of injection, the rats were killed by a blow to the head. The thoracic aorta was removed and immediately placed in a Krebs-Ringer solution (composition, mM: NaCI 118, KC1 4.7, CaC12 2.5, K H 2 P O 4 1.2, MgC12 1.2, glucose 10 and N a H C O 3 25). Excess fat and connective tissue were removed and helical strips (2 × 15 mm) were then prepared (Furchgott and Bhadrakom, 1953). Each strip was suspended vertically in a 10 ml organ chamber filled with the above solution (37°C, p H 7.4) and gassed with 95% 02-5% CO 2. A force-displacement transducer was attached to each strip. After allowing the strip to reach equilibrium with a resting tension of 1 g for 1 h, the changes in isometric force were recorded on a polygraph recorder. The tissues were blotted and
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
178
2.4. Statistics
weighed at the end of the experiment. The contractile response was expressed in terms of milligrams of force per unit wet weight (milligram) of tissue.
All values are given as means _+ S.E.M. Statistical analyses were done with Tukey's method after one-way analysis of variance; a significance level of P < 0.05 was accepted. The ECs0 value (the concentration producing a half maximal response) was determined graphically from the individual dose-response curves.
2.2. In vitro study Aortic strips from rats without pretreatment were set up as above then contracted with 80 m M KC1. The strips were washed with a Krebs-Ringer solution to restore the resting tension then were preincubated with various concentrations of aclarubicin (20, 40, 60, 80 # g / m l ) in an organ chamber for 60 rain, following which they were cumulatively .contracted with KC1 or phenylephrine. The contractile response of the strip preincubated with the same volume of 0.9% saline for 60 min was measured as a control. The contractile response for each strip was expressed as a percentage of the contraction induced by 80 m M KC1 before preincubation.
3. R e s u l t s
Figure 1 shows the effect of i.p. administration of aclarubicin (4 m g / k g body weight per day) for 5 consecutive days on the contractile responses of isolated rat aortas. The contractile responses to both KC1 and phenylephrine were reduced in aortas from the aclarubicin-treated rats compared to the saline-treated control. The ECs0 value for KC1 contracture was significantly higher in the aclarubicin-treated group than in the control group (aclarubicin group, (3.39 + 0.17) × 10 -2 M; control, (2.54 + 0.18) × 10 -2 M). The ECs0 value for phenylephrine was also significantly higher in the aclarubicin-treated group (aclarubicin group, (1.72 + 0.18) X 10 - 7 M ; control, (3.30 + 0.60) × 10 -8
2.3. Substances Aclarubicin (Yamanouchi Co., Ltd.) was dissolved in 0.99~ saline (4 m g / m l concentration) and phenylephrine (Sigma) in distilled water to give a stock solution of 10 mM. The concentration of each drug was expressed as the final concentration in the organ chamber.
M). Figure 2 shows the effect of in vitro preincubation of isolated rat aortas with various concentrations (20-80 ktg/ml) of aclarubicin on their con-
B
A 150
E 150
g 100
lOO E
E
5O
,2o 5C
0
1-0 20 30 40
KCI (mM)
60
SO
I"0 -g
10 a
i0-7
i0-6
10-s
Phenylephrine (M)
Fig. 1. Effect of i.p. injection of aclarubicin (4 mg/kg body weight per day) for 5 days on contractile responses of isolated aorta upon exposure to cumulative additions of KC1 (A) and phenylephrine (B). ((3) Control (n = 7), (zx) aclarubicin-injected (n = 7). Vertical line indicates S.E.M. Asterisks denote significant differences from the control.
1-79
B
A .--. 100
100
cO 0 0 0
! i 1-0 20 30 40
60
80
KCl (mM)
0
1~1_.~ l~_e
10-7
10-e
10-s
PhenvleDhrine (M)
Fig. 2. Effect of in vitro pretreatment of isolated aortas with various concentrations of aclarubicin (o, 0 ~tg/ml; r-l, 20 #g/rnl; A, 40 #g/rnl; O, 60 /~g/ml; X, 80 /~g/ml) on subsequent contractile responses upon exposure to cumulative additions of KCI (A) and phenylephrine (B). Vertical lines indicate S.E.M. Asterisks denote significant differences from the control (0/~g/ml of aclarubicin). n = 5-7.
tractile responses. Preincubation with aclarubicin dose dependently inhibited the subsequent contractile responses to KC1 and phenylephrine compared with the responses of the control preincubated with saline. The diminished contractile responses were not completely restored after removal of aclarubicin (80/~g/ml) (data not shown).
4. Discussion
Although the vascular effect of aclarubicin is not known, there are reports about the effects of doxorubicin on vascular tonus. It was shown that doxorubicin increased the coronary perfusion pressure of isolated dog hearts (Mhatre et al., 1971), and increased the mean arterial pressure and decreased renal blood flow in rats (Tvete et al., 1982). These results suggest that doxorubicin possesses a vasoconstrictive action. On the contrary, doxorubicin was reported to induce hypotension by release of histamine in beagle dogs (Herman et al., 1978). Thus, doxorubicin seems to produce different acute effects on vascular tonus depending on the species of animals or type of blood vessels. Recently, it has been reported that the sensitivity of the contractile response of isolated aortic strips to norepinephrine was attenuated by chronic treatment of rats with doxorubicin (Dalske and Hardy, 1988). The authors
also reported that doxorubicin in high concentration when added to the bathing medium occasionally caused a slight constriction of isolated rat aortic strips. In our experiments, in vitro preincubation of aortic strips with doxorubicin (80 /~g/ml) for 60 min did not attenuate the contractile responses to KC1 and phenylephrine (data not shown). These results suggest that the inhibitory effect of doxorubicin on vasoconstriction may be induced by its chronic action. In this study, aclarubicin was shown to attenuate aortic contractile responses in both the ex vivo and in vitro experiments. Both the sensitivity and reactivity of aortic contractile responses to KC1 and phenylephrine were diminished by treatment of rats with aclarubicin. The ex vivo experiment showed that suppression of vasoconstriction was induced by administration of aclarubicin for only 5 days. Accordingly, the vascular effect of aclarubicin may be induced by its acute action. Dalske and Hardy (1988) speculated that doxorubicin may interact with adrenoceptors on vascular smooth muscle and suppress the contractile response. However, they did not study the effect of doxorubicin administration on vascular responses not mediated by receptor stimulation. It is known that phenylephrine induces a vasocontractile response via stimulation of oq-adrenoceptors on vascular smooth muscle (Langer, 1981), on the other hand, the vasocontractile response to KC1 is not media-
180
ted by receptor stimulation but is due to opening of the voltage-dependent calcium channel by membrane depolarization (Godfraind and Kaba, 1972). Aclarubicin attenuated the contractile responses to both KC1 and phenylephrine. Thus, it is likely that aclarubicin disturbs the common contractile mechanism after receptor stimulation in vascular smooth muscle. Further experiments are required to elucidate the precise mechanism of the vascular effect of anthracycline drugs.
References Dalske, H.F. and K. Hardy, 1988, Effect of low-dose doxorubicin on calcium content and norepinephrine response in rat aorta, European J. Cancer Clin. Oncol. 24, 979. Furchgott, R.F. and S. Bhadrakom, 1953, Reactions of strips of rabbit aorta to epinephrine, isopropylarterenol, sodium nitrite and other drugs, J. Pharmacol. Exp. Ther. 108, 129. Godfraind, T. and A. Kaba, 1972, The role of calcium in the
action of drugs on vascular smooth muscle, Arch. Int. Pharmacodyn. Ther. 196 (Suppl.), 35. Herman, E., R. Young and S. Krop, 1978, Doxorubicin-induced hypotension in the beagle dog, Agents Actions 8, 551. Kantrowitz, N.E. and M.R. Bristow, 1984, Cardiotoxicity of antitumor agents, Prog. Cardiovasc. Dis. 27, 195. Langer, S.Z., 1981, Presynaptic regulation of the release of catecholamines, Pharmacol. Rev. 32, 337. Mhatre, R., E. Herman, A. Huidobro and V. Waravdekar, 1971, The possible relationship between metabolism and cardiac toxicity of daunomycin and related compounds, J. Pharmacol. Exp. Ther. 178, 216. Oki, T., Y. Matsuzawa, A. Yoshimoto, K. Numata, I. Kitamura, S. Hori, A. Takamatsu, H. Umezawa, M. Ishizuka, H. Naganawa, H. Suda, M. Hamada and T. Takeuchi, 1975, New antitumor antibiotics, aclacinomycins A and B, J. Antibiot. 28, 830. Tvete, S., A. Hope, E.A. Elsayed and G. Clausen, 1982, Effect of adriamycin on blood flow in renal tumor and normal renal tissue, European J. Cancer Clin. Oncol. 18, 565. Wakabayashi, T., T. Oki, H. Tone, S. Hirano and K. Omori, 1980, A comparative electron microscopic study of aclacinomycin and adriamycin cardiotoxicities in rabbits and hamsters, J. Electron Microsc. 29, 106.
177
EJP 20442 Short communication
Effect of aclarubicin on contractile response of rat aorta I c h i r o W a k a b a y a s h i *, K a t s u h i k o H a t a k e a a n d E i z o K a k i s h i t a Second Department of lnternal Medicine, and 1 Department of Legal Medicine, Hyogo Collegeof Medicine, Hyogo, 663, Japan
Received 6 June 1989, accepted 20 June 1989
The effect of aclarubicin on vasocontractility was investigated using aortic strips isolated from rats. The aortic strips from rats injected i.p. with aclarubicin (4 mg/kg body weight per day for 5 consecutive days) showed diminished contractile responses to KC1 and phenylephrine in comparison with the controls injected with 0.9% saline. In vitro preincubation of rat aorta with aclarubicin (20-80 #g/ml) attenuated the aortic contractile responses to KC1 and phenylephrine compared with the control preincubated with 0.9% saline. These results suggest that aclarubicin reduces the contractility of vascular smooth muscle directly. Aclarubicin; Anthracycline antibiotics; Vasoconstriction
1. Introduction Anthracycline derivatives have a potent antineoplastic action, however their cardiotoxicity limits their utility for the treatment of neoplastic diseases (Kantrowitz and Bristow, 1984). Aclarubicin is an anthracycline derivative isolated from Streptomyces galilaeus (Old et al., 1975). It was reported that morphological changes in the myocardium of animals treated with aclarubicin are less pronounced than those in doxorubicintreated animals (Wakabayashi et al., 1980). There are many reports on the cardiac effect of anthracycline drugs, but only few reports on their vascular effect. We investigated the effect of aclarubicin on vasocontractility in ex vivo and in vitro experiments using the aortic strips isolated from rats.
* To whom all correspondence should be addressed: Second Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho,Nishinomiya, Hyogo, 663, Japan.
2. Materials and methods 2.1. E x vivo study
Male Wistar rats (400-450 g) received an i.p. injection of 4 m g / k g body weight per day of aclarubicin diluted with 0.9% sterile saline for 5 consecutive days. Control rats received Lp. the same volume of 0.9% sterile saline. On the 6th day after the start of injection, the rats were killed by a blow to the head. The thoracic aorta was removed and immediately placed in a Krebs-Ringer solution (composition, mM: NaCI 118, KC1 4.7, CaC12 2.5, K H 2 P O 4 1.2, MgC12 1.2, glucose 10 and N a H C O 3 25). Excess fat and connective tissue were removed and helical strips (2 × 15 mm) were then prepared (Furchgott and Bhadrakom, 1953). Each strip was suspended vertically in a 10 ml organ chamber filled with the above solution (37°C, p H 7.4) and gassed with 95% 02-5% CO 2. A force-displacement transducer was attached to each strip. After allowing the strip to reach equilibrium with a resting tension of 1 g for 1 h, the changes in isometric force were recorded on a polygraph recorder. The tissues were blotted and
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
178
2.4. Statistics
weighed at the end of the experiment. The contractile response was expressed in terms of milligrams of force per unit wet weight (milligram) of tissue.
All values are given as means _+ S.E.M. Statistical analyses were done with Tukey's method after one-way analysis of variance; a significance level of P < 0.05 was accepted. The ECs0 value (the concentration producing a half maximal response) was determined graphically from the individual dose-response curves.
2.2. In vitro study Aortic strips from rats without pretreatment were set up as above then contracted with 80 m M KC1. The strips were washed with a Krebs-Ringer solution to restore the resting tension then were preincubated with various concentrations of aclarubicin (20, 40, 60, 80 # g / m l ) in an organ chamber for 60 rain, following which they were cumulatively .contracted with KC1 or phenylephrine. The contractile response of the strip preincubated with the same volume of 0.9% saline for 60 min was measured as a control. The contractile response for each strip was expressed as a percentage of the contraction induced by 80 m M KC1 before preincubation.
3. R e s u l t s
Figure 1 shows the effect of i.p. administration of aclarubicin (4 m g / k g body weight per day) for 5 consecutive days on the contractile responses of isolated rat aortas. The contractile responses to both KC1 and phenylephrine were reduced in aortas from the aclarubicin-treated rats compared to the saline-treated control. The ECs0 value for KC1 contracture was significantly higher in the aclarubicin-treated group than in the control group (aclarubicin group, (3.39 + 0.17) × 10 -2 M; control, (2.54 + 0.18) × 10 -2 M). The ECs0 value for phenylephrine was also significantly higher in the aclarubicin-treated group (aclarubicin group, (1.72 + 0.18) X 10 - 7 M ; control, (3.30 + 0.60) × 10 -8
2.3. Substances Aclarubicin (Yamanouchi Co., Ltd.) was dissolved in 0.99~ saline (4 m g / m l concentration) and phenylephrine (Sigma) in distilled water to give a stock solution of 10 mM. The concentration of each drug was expressed as the final concentration in the organ chamber.
M). Figure 2 shows the effect of in vitro preincubation of isolated rat aortas with various concentrations (20-80 ktg/ml) of aclarubicin on their con-
B
A 150
E 150
g 100
lOO E
E
5O
,2o 5C
0
1-0 20 30 40
KCI (mM)
60
SO
I"0 -g
10 a
i0-7
i0-6
10-s
Phenylephrine (M)
Fig. 1. Effect of i.p. injection of aclarubicin (4 mg/kg body weight per day) for 5 days on contractile responses of isolated aorta upon exposure to cumulative additions of KC1 (A) and phenylephrine (B). ((3) Control (n = 7), (zx) aclarubicin-injected (n = 7). Vertical line indicates S.E.M. Asterisks denote significant differences from the control.
1-79
B
A .--. 100
100
cO 0 0 0
! i 1-0 20 30 40
60
80
KCl (mM)
0
1~1_.~ l~_e
10-7
10-e
10-s
PhenvleDhrine (M)
Fig. 2. Effect of in vitro pretreatment of isolated aortas with various concentrations of aclarubicin (o, 0 ~tg/ml; r-l, 20 #g/rnl; A, 40 #g/rnl; O, 60 /~g/ml; X, 80 /~g/ml) on subsequent contractile responses upon exposure to cumulative additions of KCI (A) and phenylephrine (B). Vertical lines indicate S.E.M. Asterisks denote significant differences from the control (0/~g/ml of aclarubicin). n = 5-7.
tractile responses. Preincubation with aclarubicin dose dependently inhibited the subsequent contractile responses to KC1 and phenylephrine compared with the responses of the control preincubated with saline. The diminished contractile responses were not completely restored after removal of aclarubicin (80/~g/ml) (data not shown).
4. Discussion
Although the vascular effect of aclarubicin is not known, there are reports about the effects of doxorubicin on vascular tonus. It was shown that doxorubicin increased the coronary perfusion pressure of isolated dog hearts (Mhatre et al., 1971), and increased the mean arterial pressure and decreased renal blood flow in rats (Tvete et al., 1982). These results suggest that doxorubicin possesses a vasoconstrictive action. On the contrary, doxorubicin was reported to induce hypotension by release of histamine in beagle dogs (Herman et al., 1978). Thus, doxorubicin seems to produce different acute effects on vascular tonus depending on the species of animals or type of blood vessels. Recently, it has been reported that the sensitivity of the contractile response of isolated aortic strips to norepinephrine was attenuated by chronic treatment of rats with doxorubicin (Dalske and Hardy, 1988). The authors
also reported that doxorubicin in high concentration when added to the bathing medium occasionally caused a slight constriction of isolated rat aortic strips. In our experiments, in vitro preincubation of aortic strips with doxorubicin (80 /~g/ml) for 60 min did not attenuate the contractile responses to KC1 and phenylephrine (data not shown). These results suggest that the inhibitory effect of doxorubicin on vasoconstriction may be induced by its chronic action. In this study, aclarubicin was shown to attenuate aortic contractile responses in both the ex vivo and in vitro experiments. Both the sensitivity and reactivity of aortic contractile responses to KC1 and phenylephrine were diminished by treatment of rats with aclarubicin. The ex vivo experiment showed that suppression of vasoconstriction was induced by administration of aclarubicin for only 5 days. Accordingly, the vascular effect of aclarubicin may be induced by its acute action. Dalske and Hardy (1988) speculated that doxorubicin may interact with adrenoceptors on vascular smooth muscle and suppress the contractile response. However, they did not study the effect of doxorubicin administration on vascular responses not mediated by receptor stimulation. It is known that phenylephrine induces a vasocontractile response via stimulation of oq-adrenoceptors on vascular smooth muscle (Langer, 1981), on the other hand, the vasocontractile response to KC1 is not media-
180
ted by receptor stimulation but is due to opening of the voltage-dependent calcium channel by membrane depolarization (Godfraind and Kaba, 1972). Aclarubicin attenuated the contractile responses to both KC1 and phenylephrine. Thus, it is likely that aclarubicin disturbs the common contractile mechanism after receptor stimulation in vascular smooth muscle. Further experiments are required to elucidate the precise mechanism of the vascular effect of anthracycline drugs.
References Dalske, H.F. and K. Hardy, 1988, Effect of low-dose doxorubicin on calcium content and norepinephrine response in rat aorta, European J. Cancer Clin. Oncol. 24, 979. Furchgott, R.F. and S. Bhadrakom, 1953, Reactions of strips of rabbit aorta to epinephrine, isopropylarterenol, sodium nitrite and other drugs, J. Pharmacol. Exp. Ther. 108, 129. Godfraind, T. and A. Kaba, 1972, The role of calcium in the
action of drugs on vascular smooth muscle, Arch. Int. Pharmacodyn. Ther. 196 (Suppl.), 35. Herman, E., R. Young and S. Krop, 1978, Doxorubicin-induced hypotension in the beagle dog, Agents Actions 8, 551. Kantrowitz, N.E. and M.R. Bristow, 1984, Cardiotoxicity of antitumor agents, Prog. Cardiovasc. Dis. 27, 195. Langer, S.Z., 1981, Presynaptic regulation of the release of catecholamines, Pharmacol. Rev. 32, 337. Mhatre, R., E. Herman, A. Huidobro and V. Waravdekar, 1971, The possible relationship between metabolism and cardiac toxicity of daunomycin and related compounds, J. Pharmacol. Exp. Ther. 178, 216. Oki, T., Y. Matsuzawa, A. Yoshimoto, K. Numata, I. Kitamura, S. Hori, A. Takamatsu, H. Umezawa, M. Ishizuka, H. Naganawa, H. Suda, M. Hamada and T. Takeuchi, 1975, New antitumor antibiotics, aclacinomycins A and B, J. Antibiot. 28, 830. Tvete, S., A. Hope, E.A. Elsayed and G. Clausen, 1982, Effect of adriamycin on blood flow in renal tumor and normal renal tissue, European J. Cancer Clin. Oncol. 18, 565. Wakabayashi, T., T. Oki, H. Tone, S. Hirano and K. Omori, 1980, A comparative electron microscopic study of aclacinomycin and adriamycin cardiotoxicities in rabbits and hamsters, J. Electron Microsc. 29, 106.