Acta Physzol Scund 1991, 141, 141-142
Dactinomycin inhibits non-cholinergic bronchoconstriction by capsaicin-sensitive sensory nerves in the guinea-pig by antagonizing neurokinin 2 receptor activation J. M. L U N D B E R G and Y.-P. LOU Department of Pharmacology, Karolinska Institute, 104 01 Stockholm, Sweden
Substance P (SP) analogues with D-amino acid substitutions inhibit the SP, capsaicin and electrical field stimulation (EFS) evoked non-adrenergic, noncholinergic (NANC) bronchoconstriction in the guinea-pig suggesting that SP was the mediator of these responses (Lundberg et al. 1983). These peptide analogues also attenuated the potent bronchoconstrictor effects of the costored tachykinin neurokinin A (NKA) (Karlsson & Persson 1985), which complicated the interpretation of the data, however. At present at least three distinct tachykinin receptors have been characterized and designed neurokinin 1 (NKl), neurokinin 2 (NK2) and neurokinin 3 (NK3) receptors (Regoli et al. 1988). SP has the highest affinity for NK1-, NKA for NK2- and neurokinin B (NKB) for NK3- receptors. The peptide analogues Sar 9, Met 11 (0,)-SP (Nle 10)-NKA (+lo) and Pro 7-NKB have been demonstrated to have more selective actions than the endogenous peptides at NK1-, NK2and NK3-receptors, respectively (Regoli et al. 1988). McKnight et al. 1988 recently reported that cylization of certain peptide analogues with tachykinin antagonistic activity selectively enhanced the affinity for the NK2 receptor. We have therefore studied the possible influence on tachykinin responses of cyclic peptides especially with D-aminoacid substitutions. In the present study we report that Dactinomycin, an oncolyticantibiotic isolated from streptomyces species, which contains a central phenoxazone ring with two cyclic peptides (Thr-D-Val-Pro-Sar-MeVal) and classically binds to DNA and inhibits protein synthesis (Glaubiger & Ramu, 1982), can be used to selectively inhibit NK2-receptor activation. Guinea-pig hilus bronchi were mounted on two L-
Received 27 September 1990, accepted 26 September 1990. Key mords : capsaicin, dactinomycin, neurokinin A, neurokinin 2 receptors, pulmonary afferents. Correspondence : Jan M. Lundberg, Department of Pharmacology, Karolinska Institute, Box 60400, 104 01 Stockholm, Sweden.
shape holders and contractile tension was registered in vitro. EFS ( 5 Hz, 1 msec for 10 s) was performed in the presence of atropine M) to elicit NANC M) tachyphylaxis contractions or after capsaicin ( to study cholinergic (atropine sensitive responses), Capsaicin (3.10-RM), SP (3.10-' M), Sar 9, Met 11 (0,)-SP (3.10P M), NKA (lo-' M) or (Nle 10)-NKA ( 4 1 0 ) (3.10-RM) were added into the organ baths. The concentrations were chosen to cause contractions of similar magnitudes as the EFS responses. The electrical stimulations or addition of agents were performed in the absence or after preincubation with DAC (10 M or 5.10P M) for 15 min. The following sources of drugs were used: SP, NKA (Nle 10)-NKA (+lo), Sar 9, Met 11 (0,)-SP and Pro-7 NKB (Peninsula, USA), capsaicin (Fluka, Switzerland) and DAC (Sigma, USA). DAC did not influence basal bronchial tone per se. The contractions evoked by low concentrations of NKA and (Nle 10) NKA (4-10) were totally blocked by DLIC M, while the SP or Sar 9, Met 11 (0,)-SP responses were not influenced even by DAC 5.10-5 M (Fig. 1). Pro-7 (NKB) did not contract the bronchi in concentrations up to lo-' M. The NANC bronchoconstriction evoked by EFS was reduced by 60% at 10-5 M DAC and by 75% at 5.10P M DAC (Fig. 1). The cholinergic contractile response to EFS was not influenced by DAC 10-5 or 5.10-5 M, however (Fig. 1). The capsaicin contraction was reduced by 82% after preincubation with DAC 5.10-' M (Fig. 1). The present data show that DAC inhibited the NKA and Nle 10 (NKA 4 1 0 ) but not the SP or Sar 9, Met 11 (0,) SP evoked bronchoconstriction implying that DAC served as a selective NKA-2 antagonist possibly due to its cyclic peptide components (see McKnight et al. 1988). In contrast to earlier ' tachykinin' antagonists, DAC did not evoke any bronchoconstriction per se which makes it more convenient to use (see Lundberg et al. 1983). Considering the marked inhibition by DAC of the EFS or capsaicin evoked bronchoconstriction the receptors close to release sites of endogenous tachykinins are likely to be mainly of the NK2 type.
141
142
3. M. Lundberg 1201
and Y.-P. Lou T
The present study was supported by grants from the Swedish MRC (14X-6554), the American Council for Tobacco Research, the Swedish Tobacco Company, the Swedish Work and Environmental fund and the Swedish Environmental Protection Board.
REFERENCES GLAUBIGER, D. & RAMU, A. 1982. Antitumour antibiotics. In: B. A. Crabner (ed.) Pharmacological Principles of Cancer Treatment, pp. 402415. W.B. Saunders, Philadelphia. " KARLSSON, J.A. & PERSSON, C.G.A. 1985. Effects of NKA NKA "c Cap SP SP Chol different substance P analogs on tachykinin induced (4-1 0) (S9Mll) contraction of airway smooth muscle. In: R. Fig. 1. Contractile responses on isolated guinea-pig Hikansson and F. Sundler (eds.) Tachykinin Anthilus bronchi by neurokinin A, NKA (lo-' M) (Nle agonists, pp. 181-188. Elsevier, Amsterdam. 10)-NKA (+lo), (3.10-' M), NANC = non-adren- LUNDBERG, J.M., SARIA, A,, BRODIN, E., ROSELL, S. & ergic, non-cholinergic electrical field stimulation FOLKERS, K. 1983. A substance P antagonist inhibits M), capresponse in the presence of atropine ( vagally induced inflammation and bronchial smooth saicin, (cap 3.10-' M), substance P (SP, 3.10-* M). Sar muscle contraction in the guinea pig. Proc Natl 9Met 11 (O,)SP(S9 M11 SP,3.10-8~)orcholinergic Acad Sci, USA, 80, 1120-1 124. phylaxis to capsaicin M). Data are given as MCKNIGHT,A.T., MAGUNI,J.J., WILLIAMS,B.J., means+SEM (n = 6) and expressed as percent of FOSTER, A.C., TRIDGETT, R. & IVERSEN, L.L. 1988. control responses compared to in the presence of Pharmacological specificity of synthetic peptides as Dactinomycin M) for NKA, (Nle 10)-NKA antagonists at tachykinin receptors. Regulatory ( 4 1 0 ) or 5.10-5 M for SP, Sar 9, Met 11 (0,)SP, Peptides 22, 127. capsaicin and the NANC and cholinergic nerve REGOLI,D., DRAPEAU, G., DION, S., COUTURE, R. responses. *** P < 0.001, Student's t-test. 1988. New selective agonists for neurokinin receptors : pharmacological tools for receptor characterization. Trends in Pharmacol Sci 9, 290-295.
Dactinomycin inhibits non-cholinergic bronchoconstriction by capsaicin-sensitive sensory nerves in the guinea-pig by antagonizing neurokinin 2 receptor activation J. M. L U N D B E R G and Y.-P. LOU Department of Pharmacology, Karolinska Institute, 104 01 Stockholm, Sweden
Substance P (SP) analogues with D-amino acid substitutions inhibit the SP, capsaicin and electrical field stimulation (EFS) evoked non-adrenergic, noncholinergic (NANC) bronchoconstriction in the guinea-pig suggesting that SP was the mediator of these responses (Lundberg et al. 1983). These peptide analogues also attenuated the potent bronchoconstrictor effects of the costored tachykinin neurokinin A (NKA) (Karlsson & Persson 1985), which complicated the interpretation of the data, however. At present at least three distinct tachykinin receptors have been characterized and designed neurokinin 1 (NKl), neurokinin 2 (NK2) and neurokinin 3 (NK3) receptors (Regoli et al. 1988). SP has the highest affinity for NK1-, NKA for NK2- and neurokinin B (NKB) for NK3- receptors. The peptide analogues Sar 9, Met 11 (0,)-SP (Nle 10)-NKA (+lo) and Pro 7-NKB have been demonstrated to have more selective actions than the endogenous peptides at NK1-, NK2and NK3-receptors, respectively (Regoli et al. 1988). McKnight et al. 1988 recently reported that cylization of certain peptide analogues with tachykinin antagonistic activity selectively enhanced the affinity for the NK2 receptor. We have therefore studied the possible influence on tachykinin responses of cyclic peptides especially with D-aminoacid substitutions. In the present study we report that Dactinomycin, an oncolyticantibiotic isolated from streptomyces species, which contains a central phenoxazone ring with two cyclic peptides (Thr-D-Val-Pro-Sar-MeVal) and classically binds to DNA and inhibits protein synthesis (Glaubiger & Ramu, 1982), can be used to selectively inhibit NK2-receptor activation. Guinea-pig hilus bronchi were mounted on two L-
Received 27 September 1990, accepted 26 September 1990. Key mords : capsaicin, dactinomycin, neurokinin A, neurokinin 2 receptors, pulmonary afferents. Correspondence : Jan M. Lundberg, Department of Pharmacology, Karolinska Institute, Box 60400, 104 01 Stockholm, Sweden.
shape holders and contractile tension was registered in vitro. EFS ( 5 Hz, 1 msec for 10 s) was performed in the presence of atropine M) to elicit NANC M) tachyphylaxis contractions or after capsaicin ( to study cholinergic (atropine sensitive responses), Capsaicin (3.10-RM), SP (3.10-' M), Sar 9, Met 11 (0,)-SP (3.10P M), NKA (lo-' M) or (Nle 10)-NKA ( 4 1 0 ) (3.10-RM) were added into the organ baths. The concentrations were chosen to cause contractions of similar magnitudes as the EFS responses. The electrical stimulations or addition of agents were performed in the absence or after preincubation with DAC (10 M or 5.10P M) for 15 min. The following sources of drugs were used: SP, NKA (Nle 10)-NKA (+lo), Sar 9, Met 11 (0,)-SP and Pro-7 NKB (Peninsula, USA), capsaicin (Fluka, Switzerland) and DAC (Sigma, USA). DAC did not influence basal bronchial tone per se. The contractions evoked by low concentrations of NKA and (Nle 10) NKA (4-10) were totally blocked by DLIC M, while the SP or Sar 9, Met 11 (0,)-SP responses were not influenced even by DAC 5.10-5 M (Fig. 1). Pro-7 (NKB) did not contract the bronchi in concentrations up to lo-' M. The NANC bronchoconstriction evoked by EFS was reduced by 60% at 10-5 M DAC and by 75% at 5.10P M DAC (Fig. 1). The cholinergic contractile response to EFS was not influenced by DAC 10-5 or 5.10-5 M, however (Fig. 1). The capsaicin contraction was reduced by 82% after preincubation with DAC 5.10-' M (Fig. 1). The present data show that DAC inhibited the NKA and Nle 10 (NKA 4 1 0 ) but not the SP or Sar 9, Met 11 (0,) SP evoked bronchoconstriction implying that DAC served as a selective NKA-2 antagonist possibly due to its cyclic peptide components (see McKnight et al. 1988). In contrast to earlier ' tachykinin' antagonists, DAC did not evoke any bronchoconstriction per se which makes it more convenient to use (see Lundberg et al. 1983). Considering the marked inhibition by DAC of the EFS or capsaicin evoked bronchoconstriction the receptors close to release sites of endogenous tachykinins are likely to be mainly of the NK2 type.
141
142
3. M. Lundberg 1201
and Y.-P. Lou T
The present study was supported by grants from the Swedish MRC (14X-6554), the American Council for Tobacco Research, the Swedish Tobacco Company, the Swedish Work and Environmental fund and the Swedish Environmental Protection Board.
REFERENCES GLAUBIGER, D. & RAMU, A. 1982. Antitumour antibiotics. In: B. A. Crabner (ed.) Pharmacological Principles of Cancer Treatment, pp. 402415. W.B. Saunders, Philadelphia. " KARLSSON, J.A. & PERSSON, C.G.A. 1985. Effects of NKA NKA "c Cap SP SP Chol different substance P analogs on tachykinin induced (4-1 0) (S9Mll) contraction of airway smooth muscle. In: R. Fig. 1. Contractile responses on isolated guinea-pig Hikansson and F. Sundler (eds.) Tachykinin Anthilus bronchi by neurokinin A, NKA (lo-' M) (Nle agonists, pp. 181-188. Elsevier, Amsterdam. 10)-NKA (+lo), (3.10-' M), NANC = non-adren- LUNDBERG, J.M., SARIA, A,, BRODIN, E., ROSELL, S. & ergic, non-cholinergic electrical field stimulation FOLKERS, K. 1983. A substance P antagonist inhibits M), capresponse in the presence of atropine ( vagally induced inflammation and bronchial smooth saicin, (cap 3.10-' M), substance P (SP, 3.10-* M). Sar muscle contraction in the guinea pig. Proc Natl 9Met 11 (O,)SP(S9 M11 SP,3.10-8~)orcholinergic Acad Sci, USA, 80, 1120-1 124. phylaxis to capsaicin M). Data are given as MCKNIGHT,A.T., MAGUNI,J.J., WILLIAMS,B.J., means+SEM (n = 6) and expressed as percent of FOSTER, A.C., TRIDGETT, R. & IVERSEN, L.L. 1988. control responses compared to in the presence of Pharmacological specificity of synthetic peptides as Dactinomycin M) for NKA, (Nle 10)-NKA antagonists at tachykinin receptors. Regulatory ( 4 1 0 ) or 5.10-5 M for SP, Sar 9, Met 11 (0,)SP, Peptides 22, 127. capsaicin and the NANC and cholinergic nerve REGOLI,D., DRAPEAU, G., DION, S., COUTURE, R. responses. *** P < 0.001, Student's t-test. 1988. New selective agonists for neurokinin receptors : pharmacological tools for receptor characterization. Trends in Pharmacol Sci 9, 290-295.
![Selective inhibition by dactinomycin of NANC sensory bronchoconstriction and [125I]NKA binding due to NK-2 receptor antagonism.](/assets/img/hold.png)
