183

European Journal of Pharmacology, 194 (1991) 183-188 © 1991 Elsevier Science Publishers B.V. 0014-2999/91/$03.50 ADONIS 0014299991002038

EJP 51752

Effects of actinomycin D on airway constriction induced by tachykinins and capsaicin in guinea-pigs T a k a s h i Fujii, M a s a k o Murai, H i r o s h i M o r i m o t o , M o t o a k i N i s h i k a w a 1 a n d S u m i o K i y o t o h 1 Department of Pharmacology, New Drug Research Laboratories, FujisawaPharmaceutical Co., Ltd., 1-6, 2-chome, Kashima, Yodogawa-ku, Osaka 532, Japan and 1 Exploratory Research Laboratories, FufisawaPharmaceutical Co., Ltd., 2-3, 5-chome, Tokodai, Tsukuba-shi, Ibaragi 300-26, Japan Received 6 November 1990, revised MS received 4 December 1990, accepted 11 December 1990

The effects of actinomycin D on airway constriction induced by tachykinins was studied in guinea-pigs in vitro and in vivo. Actinomycin D significantly inhibited the constriction of isolated guinea-pig trachea induced by neurokinin A (NKA) and eledoisin. Conversely, substance P (SP)- and physalaemine-induced constrictions were not affected by actinomycin D. The same selectivity in the inhibitory action of actinomycin D against tachykinins was also observed in in vivo. Actinomycin D given i.v. specifically inhibited the increase in airway resistance induced by NKA. I.v. injection of NKA caused not only airway constriction but also transient systemic hypotension. Interestingly, actinomycin D injected i.v. inhibited only airway constriction and the systemic hypotension induced by NKA was not affected. These results dearly suggest that actinomycin D specifically inhibits NKA-induced airway constriction in guinea-pigs. Actinomycin D also had an inhibitory action on the airway constriction induced by capsaicin. In the case of capsaicin-induced constriction, actinomycin D was more effective on the later phase of constriction than on the acute phase. The airway constriction induced by capsaicin is thought to be mediated by the release of SP and NKA from sensory nerve endings, and the persistent increase in airway resistance induced by capsaicin is thought to be due mainly to NKA. Actinomycin D; Neurokinin A; Substance P; Airway constriction

I. Introduction

Substance P (SP), identified as an undecapeptide by Chang and Leaman (1970), is a putative neurotransmitter found in both peripheral and central neurons. Substance P has numerous physiological actions, e.g. it causes hypotension as a result of vasodilatation, increases vascular permeability, contracts smooth muscle cells (ileum, trachea, etc.), stimulates salivary glands, and is involved in pain generation and inflammation (Pernow, 1983). In 1983 other mammalian tachykinins, (neurokinins), neurokinin A (NKA) and neurokinin B (NKB), were discovered (Kimura et al., 1983; Kanagawa et al., 1983) and the co-existence of N K A with SP in the same neuron was demonstrated (Nawa et al., 1983; Hua et al., 1985). The airways are well innervated with capsaicin-sensitive primary afferents which contain neurokinins as

Correspondence to: T. Fujii, Department of Pharmacology, New Drug Research Laboratories, Fujisawa PharmaceUtical Co., Ltd., 1-6, 2-chome, Kashima, Yodogawa-ku, Osaka 532, Japan.

neurotransmitter (Lundberg et al., 1984). Non-specific stimulation of the airway causes the release of neurokinins followed by marked bronchoconstriction, airway edema and hypersecretion of mucus (Lundberg and Saria, 1982a,b; Morimoto et al., 1989). Since the effects induced by neurokinins in airways resemble the typical pathological features of asthma, there has been considerable interest in the possible involvement of neurokinins in airway pathophysiology (Barnes et al., 1988). Although SP and N K A are released from the same neuron simultaneously, both neuropeptides have different effects on airways. For airway constriction, N K A is more potent than SP while SP is more potent at inducing airway edema than NKA. Based on the different physiological effects of each neurokinin, the existence of three receptors for neurokinins has been suggested (Buck and Butcher, 1986; Regoli et al., 1987). Althought sensory neurokinins have several effects which suggest they have an important role in asthma, direct evidence for their involvement is still lacking because there are no satisfactory selective antagonists that can be used clinically or experimentally. We now report on the antagonistic actions of the antibiotic actinomycin D on the airway constriction induced by tachykinins.

184 2. M a t e r i a l s a n d m e t h o d s

2.1. Guinea-pig tracheal constriction in vivo Male albino guinea-pigs weighing 300-400 g were stunned and bled. The trachea were rapidly removed and a zigzag strip preparation of trachea was placed in oxygenated (95% 02 and 5% CO2) standard Tyrode solution under a resting tension of 0.5 g. After a 60-min equilibration period, the response to each agonist was recorded 2-3 times to get a stable contraction response. The agonists used and their final concentrations were as follows: N K A ( 1 0 - 9 - 1 0 - 8 M), SP (10 - 7 M), eledoisin (10 -8 M), physalaemine (10 -7 M), acetylcholine (10 -6 M). For the experiments involving pretreatment with agonist, the test drug was added after the m a x i m u m construction was obtained by addition of each agonist and relaxation was recorded after 10 min. All experiments were carried out in the presence of indomethacin (5.6 x 10 -6 M) to prevent prostaglandin-mediated relaxation. For the experiments involving pretreatment with test drugs, the stable constriction was used as control. After the preparation had been washed, the test drug was added and 10 min later, constriction was induced with the same agonist. This response was then compared to the control response.

2.3. Materials The drugs used were as follows: actinomycin D (Sigma), adriamycin (Kyowa Hakkou), quinacrine (Nakarai), N K A (Cambridge Research Biochemical), SP (Tanpaku-ken), eledoisin (Peninsula Laboratories), physalaemine ( C a m b r i d g e Research Biochemical), capsaicin (Sigma), acetylcholin (Daiichi Seiyaku), histamine (Nakarai). 2.4. Statistical analysis Results are expressed as means + S.E. of number of experiments as indicated. Statistical analysis was done by means of Student's t-test for paired data.

3. R e s u l t s

3.1. Effects of actinomycin D on tracheal constriction in vitro 3.1.1 Agonist pretreatment Figure 1 shows a typical experimental tracing of the effect of actinomycin D on N K A - and SP-induced tracheal constriction. A c t i n o m y c i n D (10-5 M) markedly relaxed the NKA-constricted trachea but not the SP-constricted trachea. Figure 2 summarized data on the relaxing effect of actinomycin D (10 -5 M) on trachea preconstricted with various agonists. Significant relaxing effects of actinomycin D were observed only

2.2. Guinea-pig airway constriction in vivo CONSm~Te~g) Male albino guinea-pigs weighing 340-440 g were anesthetized by i.p. injection of pentobarbital (10 m g / a n i m a l ) . The jugular vein and trachea were cannulated and the animals were artificially ventilated (5 ml a i r / 6 0 stroke per min). The airway resistance was recorded by using the method of Konzett and R~Sssler (1940) with minor modification. The pressure in the respirator system, i.e. the insufflation pressure, was measured constantly with a transducer (TP 200T Nihon Kohden) connected to a polygraph (AP-601G Nihon Kohden). Each agonist was injected i.v. every 15 min through the jugular vein cannula to induce airway constriction. Agonist was administered repeatedly until a reproducible constriction was obtained (control). A test drug was then administered i.v. followed by the same agonist. The resulting constriction was compared with the control constriction. The agonists and doses used were as follows; N K A (1.1 /tg/kg), SP (13.5 # g / k g ) , capsaicin (3.1 ~ g / k g ) , histamine (8 /~g/kg). In some experiments, systemic arterial blood pressure was monitored at the same time via a catheter in a carotid artery connected to a pressure transducer and polygraph.

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ACTINOM~CIN D

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Fig. l. Typical tracing obtained after agonist pretreatment. Isolated guinea-pig trachea were constricted by neurokinin A ((A) 10 -s M) and substance P ((B) 10 - 7 M). After a stable constriction had been obtained, actinomycin D was added (10 -5 M). NKA: neurokinin A, SP: substance P.

185 TABLE 1 Effects of actinomycin D, adriamycin and quinacrine on the constriction of isolated guinea-pig trachea induced by neurokinin. Each value is the mean + S.E. Significantly different from the control, a p < 0.01.

z 50

_o i_m

Drugs

"I"

T

itll

1"

NKA ELE SP PHY Ach (10-8M) (10-8M) (IO-'M) (IO-'M) (IO"~M) Fig. 2. Effects of actinomycin D (10-5 M) on pre-constricted guineapig isolated trachea induced by various agonists. Each value is the mean + S.E. of four experiments. NKA: neurokinin A, Ele: eledoisin, SP: substance P, Phy: Physalaemine,Ach: acetylcholine. Significantly different from the control, * P < 0.05, * * P < 0.01. with trachea preconstricted with N K A (10 -8 M) and eledoisin (10 -8 M). Actinomycin D had no effects on SP (10 -7 M)-, physalaemine (10 -7 M)- and acetylcholine ( 1 0 - 6 M)-induced tracheal constriction. The doses of each agonists used were determined so as to yield the same extent of tracheal constriction. These results suggest strongly that actinomycin D is a selective inhibitor of N K A - and eledoisin-induced tracheal constriction. 3.1.2. Test drug pretreatment The dose-response curve for actinomycin D against NKA-induced tracheal constriction was studied in test drug pretreatment experiments (fig. 3). Actinomycin D inhibited 1 0 - 9 and 10 -8 M NKA-induced tracheal constriction in a dose-dependent manner. The IC50 values for actinomycin D against 10 -9 and 10 -8 M N K A were 100

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105

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CONCENTRATION OF ACTINOMYClN D (M) Fig. 3. Inhibitory effects o f actinomycin D on the constriction o f isolated guinea-pig trachea induced by various agonists after pretreatment o f the trachea with test drugs. Actinomycin D was added 10 min before agonist challenge. ( o ) N e u r o k i n i n A (10 - 9 M), (e) neurokinin A (10 - s M), (z~) substance P (10 - s M), (D) acetylcholine (10 -7 M). Each value is the mean ___S.E. o f five experiments. Sigruficantly different from the control, * P < 0.05, * * P < 0.01.

Actinomycin D Adriamycin

10 - 5 10- 4

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Quinacrine

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1.8 × 10 -6 and 2.8 × 10 -5 M, respectively. Actinomycin D did not inhibit 10 -8 M SP- and 1 0 - 7 M acetylcholine-induced tracheal constriction even in doses of 3.2 × 10 -5 M. These data show that actinomycin D also works as a selective inhibitor of N K A in drug pretreatment experiments. Actinomycin D is best known as a potent inhibitor of R N A synthesis. This effect of actinomycin D is thought to be caused by an interaction between actinomycin D and D N A (Reich et al., 1967). It is not likely that the anti-NKA effect of actinomycin D is related to inhibition of R N A synthesis. To confirm this, other R N A synthesis inhibitors, namely adriamycin and quinacrine, were tested in the same experiment. As shown in table 1, neither adriamycin ( 1 0 - 4 M) nor quinacrine (10 -5 M) had an effect on N K A ( 1 0 - 9 M)-induced tracheal constriction. 3.2. Effects of actinomycin D on airway constriction in vioo 3.2.1. Effects on NKA-, SP- and histamine-induced airway constriction in guinea-pigs Actinomycin D given i.v. 2 min prior to challenge with N K A clearly inhibited the N K A (1.1 ~ g / k g i.v.)induced increase in airway resistance in a dose-dependent manner (fig. 4). The EDs0 value for actinomycin D was 1.9 m g / k g . The increase in airway resistance induced by SP (13.5 # g / k g i.v.) and by histamine (8 ~tg/kg i.v.) was not affected by the treatment with actinomycin D, even at a dose of 10 m g / k g . These results show that actinomycin D is also effective in vivo and that the inhibitory action of actinomycin D is selective, which was also suggested by the results of the in vitro experiments. The a n t i - N K A effects of actinomycin D in vivo were relatively short-lasting since the anti-NKA activity of actinomycin D was hardly detectable 30-40 min after injection (data not shown). The doses of each agonist were such that the same extent of airway constriction would be induced. In some experiments, the blood pressure was monitored simultaneously, and a typical tracing is shown in fig. 5. N K A injected i.v. caused not only airway constriction but also transient systemic hypotension. Interestingly, actino-

186 100

AIRWAY RESISTANCE(cmH20) 50

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Fig. 4. Effects of actinomycin D on guinea pig airway constriction induced by neurokinin A, substance P and histamine. Actinomycin D was given i.v. 2 min before the agonist challenge. (©) Neurokinin A (1.1 #g/kg), (A) substance P (13.5 /~g/kg), ([]) histamine (8/~g/kg). Each value is the mean ± S.E. of five animals. Significantly different from the control, * * P < 0.01.

m y c i n D significantly i n h i b i t e d a i r w a y c o n s t r i c t i o n b u t d i d not affect the h y p o t e n s i o n i n d u c e d b y N K A .

3.2.2. Effects on capsaicin-induced airway constriction in guinea-pigs Capsaicin, a p u n g e n t agent i s o l a t e d f r o m r e d p e p p e r , is k n o w n to s t i m u l a t e sensory n e u r o n C - f i b e r s a n d to i n d u c e the release of n e u r o p e p t i d e s , e.g. SP a n d N K A , f r o m nerve endings (Buck a n d Burks, 1986). C a p s a i c i n injected i.v. causes a m a r k e d increase in a i r w a y resistance that is c o n s i d e r e d to be d u e to n e u r o k i n i n release ( L u n d b e r g a n d Saria, 1982a). A c t i n o m y c i n D injected i.v. 2 min b e f o r e c a p s a i c i n significantly i n h i b i t e d the

~A

A

~'rlNOM'~CIN D Fig. 5. Typical tracing of airway constriction and systemic hypotension induced by i.v. injection of neurokinin A in guinea-pigs. Airway resistance and blood pressure were monitored simultaneously in the same animal. (T) Injection of neurokinin A (1.1/tg/kg), (T) injection of actinomycin D (10 mg/kg).

increase in a i r w a y resistance (fig. 6). T h e p a t t e r n of i n h i b i t o r y effects o f a c t i n o m y c i n D on the capsaicin-ind u c e d a i r w a y c o n s t r i c t i o n h a d s o m e interesting features. A l t h o u g h a c t i n o m y c i n D i n h i b i t e d the m a x i m u m increase in a i r w a y resistance b y o n l y 30% c o m p a r e d to control, even at a d o s e of 10 m g / k g , the later p h a s e of a i r w a y c o n s t r i c t i o n was affected to a greater extent b y t r e a t m e n t with a c t i n o m y c i n D. A s shown in fig. 6, the increase in a i r w a y resistance r e a c h e d a m a x i m u m at a r o u n d 15 s after the injection of c a p s a i c i n a n d then g r a d u a l l y d e c r e a s e d to n o r m a l levels in the controls. F o u r m i n u t e s after the c a p s a i c i n t r e a t m e n t , 40-50% of

"IJNCREASE OF AIRWAY RESISTANCE C O I ~ R E D TO THE MAXIMUM INCFEASE OF CONTROL(IO0%) 100'

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Fig. 6. Effects of actinomycin D on the airway constriction induced by capsaicin in guinea-pigs. Actinomycin D ((A) 0.1 mg/kg, (B) 1 mg/kg, (C) 10 mg/kg) was given i.v. 2 min before capsaicin (3.1 /.tg/kg) challenge. (