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Contractile effects caused by thaliporphine in the guinea-pig ileum
European Journal of Pharmacology, 234 (1993) 121-123 © 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$06.00
121
EJP 21212
Short communication
Contractile effects caused by thaliporphine in the guinea-pig ileum S h e u - M e e i Yu, S h o e i - S h e n g L e e a, H w a C h o u a a n d C h e - M i n g T e n g Pharmacological Institute and a School of Pharmacy, College of Medicine, National Taiwan UniL,ersity, Taipei, Taiwan Received 9 November 1992, revised MS received 20 January 1993, accepted 2 February 1993
The intestinal effects of thaliporphine, a potent vasoconstrictor, were studied using the isolated guinea-pig ileum. Thaliporphine (0.1-100/zM) caused contraction in a concentration-dependent manner. The contraction was not affected by pretreatment of the ileum with tetrodotoxin, phentolamine, prazosin, propranolol, naloxone, atropine, diphenhydramine, methysergide, indomethacin or staurosporine. However, the contraction was inhibited markedly by nifedipine and verapamil. These results suggest that thaliporphine causes contraction of intestinal smooth muscle by a direct effect on muscle mediated by an increased Ca 2+ influx through voltage-dependent Ca 2+ channels. Contraction; Thaliporphine; Ileum (guinea-pig); Ca 2+ channels
1. Introduction
Thaliporphine, an aporphine derivative, was isolated from the plant Neolitsea konishii K (Ahmad et al., 1977) and was shown to be a potent agonist of vascular smooth muscle contractions (Teng et al., submitted). The vasoconstrictive effect of thaliporphine was abolished in the absence of extracellular Ca 2+. In addition to this effect, thaliporphine also had potent contractile activity on non-vascular tissues such as trachea, left atria and urinary bladder (unpublished observations). Recently, we found that thaliporphine increased rhythmic activity when applied cumulatively to guinea-pig ileum. In this study, we evaluated the mechanism of action of thaliporphine-induced contraction in the guinea-pig ileum.
CaC12 1.9, K H 2 P O 4 1.2, N a H C O 3 25 and glucose 11.7 mM. The Krebs solution was aerated continuously with a gas mixture of 95% 0 2 and 5% CO 2. Ileal segments (2 cm long) were mounted in an organ bath (5 ml) containing Krebs solution at 37°C. The muscle strips were initially under a resting tension of 1 g. After an equilibration period of 60 min the strips were challenged with the test drugs. Changes in tension were recorded isometrically with a Grass FT03 force-displacement transducer connected to a Grass polygraph. When the effects of antagonists were studied, the muscle strip was pretreated with each antagonist for 15 min before thaliporphine was added. Results are expressed as means + S.E.M. of tension changes (in g) for n independent experiments. Student's t-test was used to assess the significant of differences between means. A P value of < 0.05 was considered as a significant difference.
2. Materials and methods
2.1. Isolated guinea-pig ileal preparations Guinea-pigs of either sex weighing 300-400 g were killed by a blow to the head and were bled. Segments of the terminal ileum, from 2 cm of the ileo-caecal valve, were removed quickly and placed in Krebs solution composed of NaC1 118.3, KC1 4.7, MgSO 4 1.2,
Correspondence to: C.M. Teng, Pharmacological Institute, College of Medicine, National Taiwan University, No. 1, Jen-Ai Road, Section l, Taipei, Taiwan.
2.2. Drugs Thaliporphine was prepared from isoboldine, isolated from plant Neolitsea konishii K (Lee and Yang, 1992), and its purity ( > 99%) was confirmed by N M R (nuclear magnetic resonance), mass, I R (infrared) and U V (ultraviolet) spectrum. The following drugs were used: tetrodotoxin, prazosin, phentolamine, propranolol, atropine, d i p h e n h y d r a m i n e , naloxone, indomethacin, nifedipine, staurosporine and verapamil. These drugs were obtained from Sigma Chemical CO. Bay K 8644 (methyl-l,4-dihydro-2,6-dimethyl-3-nitro-4(2-trifluoromet hylphenyl)-pyridine-5-carboxylat e) was
122 TABLE 1 Influence of various pretreatments on thaliporphine (10 ~M)-induced contractions in guinea-pig ileum. Treatment
Tension (g)
Control Tetrodotoxin (1 /x M) Phentolamine (10 txM) Prazosin (0.1 /x M) Propranolol (10 tiM) Naloxone (10 p.M) Atropine (0.1 ttM) Diphenhydramine (1 t~M) Methysergide (1 /xM) Indomethacin (10 # M ) Staurosporine (1 nM) Nifedipine (0.1 /xM) Verapamil (0.3/xM)
0,75 _+0.08 0,65 4- 0.11 0.704-0.10 0.80 _+0.09 0.69+0.08 0.65_+0.07 0.624-0.06 0.78 4- 0.07 0.75 4- 0.06 0.80 4- 0.10 0.70 + 0.05 0.00 + 0.00 0.00 _+0.08
1). The concentrations of these antagonists were sufficient to block the activity of the respective agonists (data not shown). Incubation of ileum with Ca2+-free Krebs solution (containing 1 mM E G T A ) for 15 min completely abolished the thaliporphine-induced contraction. However, addition of 3 mM CaC12 at the end of the experiment restored the thaliporphine-induced contraction to the control level. Pretreatment of ileum with nifedipine (0.03 /xM) and verapamil (0.03 /xM) shifted the concentration-response curve for thaliporphine to the right. Bay K 8644 (0.1 /xM) or KCI (20 mM), added 15 min before thaliporphine was added, had no effect on the resting tension but shifted the concentration response curve for thaliporphine to the left (fig. 1).
~' P < 0.001 as compared with the control.
4. Discussion
obtained from Bayer. When drugs were dissolved in dimethylsulfoxide (DMSO), the final concentration of DMSO in the bath did not exceed 0.1% (v/v) and had no effect on the muscle contraction.
3. Results
Cumulative application of 0.1-100 /xM thaliporphine produced a concentration-dependent contraction in the guinea-pig ileum. The geometric mean of the EDs0 was 3.6 + 0.7 /xM. The contractile response of the guinea-pig ileum to thaliporphine (10/xM) was not significantly modified in the presence of tetrodotoxin (1 /xM), phentolamine (10 /xM), prazosin (0.1 /xM), propranolol (10 /xM), naloxone (10 /xM), atropine (0.1 /xM), diphenhydramine (1 /xM), methysergide (1 /xM), indomethacin (10 /xM) or staurosporine (1 nM) (table
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Thaliporphine (p.M) Fig. i. Effects of nifedipine (0.03 ~ M , o), verapamil (0.03 izM, A), Bay K 8644 (0.I ~,M, A) and KCI (20 m M , Q) on the contraction induced by cumulative application of thaliporphine in guinea-pig ileum; (o) control. Agents to be tested were added 15 rain before thaliporphine was added. Each point represents the mean + S.E.M.
(n = 6-8).
The present results demonstrate that, in the guineapig ileum, thaliporphine caused a contractile response which was not affected by tetrodotoxin. This indicates that thaliporphine exerts a direct effect on intestinal smooth muscle and that activation of sodium channels is not essential for thaliporphine to induce contraction. However, Fontaine and Lebrun (1988) found that Bay K 8644 exerted potent spasmogenic effects on the isolated mouse distal colon. The presence of tetrodotoxin lowered the threshold concentration of Bay K 8644 and enhanced the maximal responses elicited by Bay K 8644. This finding suggests that the toxin interferes with inhibitory nerves, causing constant hyperpolarization of colonic smooth muscle cells. Tetrodotoxin, by reducing the hyperpolarization mediated by the neural influence, would therefore enhance the ability of Bay K 8644 to affect voltage-sensitive Ca 2+ channels. Moreover, dihydropyridine Ca 2+ channel binding sites are rather specific for Bay K 8644 and nifedipine, and neither verapamil nor diltiazem interfere competitively with the effect of Bay K 8644 (Schramm et al., 1983). However, both nifedipine and verapamil competitively antagonized the contraction induced by thaliporphine. This indicates that different binding sites for thaliporphine and Bay K 8644 may exist on the Ca 2+ channel. It is established that the tone of ileal smooth muscle is controlled by a dense innervation and many enteric neurotransmitters. Activation of ~- or/3-adrenoceptors (Broadley and Grassby, 1985) and opiate receptors reduces the tone. In contrast, activation of acetylcholine, serotonin or histamine receptors induces contraction. In the present study, the contractile effects of thaliporphine were not affected by antagonism of aadrenoceptors (phentolamine, prazosin), /3-adrenoceptors (propranolol), opiate (naloxone), muscarinic (atropine), serotonin (methysergide) or histamine (diphen-
123
hydramine) receptors. Thus, the thaliporphine-induced response is not mediated by receptors for the wellknown contractile or relaxing neurotransmitters. Some evidence suggests that protein kinase C, which is inhibited by staurosporine (Kiyoto et al., 1987; Schachtele et al., 1988), might regulate the tonic component of receptor-mediated vascular (Abdel-Latif, 1986) and intestinal smooth muscle contractions (Holzer and Lippe, 1989). The contractile effects of thaliporphine were not affected by staurosporine, thus ruling out the involvement of protein kinase C in the thaliporphine-induced contraction. In the rat aorta, the thaliporhine-induced contraction has been shown to depend on Ca 2+ influx through voltage-dependent Ca 2+ channels. In intestinal preparations, the thaliporphine-induced contraction was inhibited by nifedipine or verapamil and potentiated by Bay K 8644 or KC1. These results suggest that the influx of extracellular Ca 2 + via voltage-dependent Ca 2 + channels is mainly responsible for the intestinal contraction. However, the underlying mechanism for direct or indirect activation of voltage-dependent Ca 2+ channels remains to be clarified.
Acknowledgements This work was supported by a Research Grant of the National Science Council of the Republic of China (NSC81-0412-B002-113).
References Abdel-Latif, A.A., 1986, Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers, Pharmacol. Rev. 38, 227. Ahmad, R., J.M. Saa and M.P. Cava, 1977, Regioselective O-demethylation in the aporphine alkaloid series, J. Org. Chem. 42, 1228. Broadley, K.J. and P.F. Grassby, 1985, Alpha- and beta-adrenoceptor-mediated responses of the guinea-pig ileum and the effects of neuronal uptake inhibition, Naunyn-Schmiedeb. Arch. Pharmacol. 331,316. Fontaine, J. and P. Lebrun, 1988, Pharmacological analysis of the effects of Bay K 8644 and organic calcium antagonists on the mouse isolated distal colon, Br. J. Pharmacol. 94, 1198. Holzer, P. and I.T. Lippe, 1989, Protein kinase C may regulate the tonic component of intestinal smooth muscle contraction in res p o n s e to substance P, Naunyn-Schmiedeb. Arch. Pharmacol. 339, 314. Kiyoto, I., S. Yamamoto, E. Aizu and R. Kato, 1987, Staurosporine, a protein kinase C inhibitor, falls to inhibit 12-O-tetradecanoylphorbol-13-acetate-caused ornithine decarboxylase induction in isolated mouse epidermal cells, Biochem. Biophys. Res. Commun. 148, 740. Lee, S.S. and H.C. Yang, 1992, Isoquinoline alkaloids from Neolitsea konishii K, J. Chin. Chem. Soc. 39, 189. Schachtele, C.R., R. Seifert and H. Osswald, 1988, Stimulus-dependent inhibition of platelet aggregation by the protein kinase C inhibitors polymyxin B, H-7 and staurosporine, Biochem. Biophys. Res. Commun. 151,542. Schramm, M., G. Thomas, R. Towart and G. Franckowiak, 1983, Novel dihydropyridines with positive inotropic action through activation of Ca 2+ channels, Nature 303, 535.
121
EJP 21212
Short communication
Contractile effects caused by thaliporphine in the guinea-pig ileum S h e u - M e e i Yu, S h o e i - S h e n g L e e a, H w a C h o u a a n d C h e - M i n g T e n g Pharmacological Institute and a School of Pharmacy, College of Medicine, National Taiwan UniL,ersity, Taipei, Taiwan Received 9 November 1992, revised MS received 20 January 1993, accepted 2 February 1993
The intestinal effects of thaliporphine, a potent vasoconstrictor, were studied using the isolated guinea-pig ileum. Thaliporphine (0.1-100/zM) caused contraction in a concentration-dependent manner. The contraction was not affected by pretreatment of the ileum with tetrodotoxin, phentolamine, prazosin, propranolol, naloxone, atropine, diphenhydramine, methysergide, indomethacin or staurosporine. However, the contraction was inhibited markedly by nifedipine and verapamil. These results suggest that thaliporphine causes contraction of intestinal smooth muscle by a direct effect on muscle mediated by an increased Ca 2+ influx through voltage-dependent Ca 2+ channels. Contraction; Thaliporphine; Ileum (guinea-pig); Ca 2+ channels
1. Introduction
Thaliporphine, an aporphine derivative, was isolated from the plant Neolitsea konishii K (Ahmad et al., 1977) and was shown to be a potent agonist of vascular smooth muscle contractions (Teng et al., submitted). The vasoconstrictive effect of thaliporphine was abolished in the absence of extracellular Ca 2+. In addition to this effect, thaliporphine also had potent contractile activity on non-vascular tissues such as trachea, left atria and urinary bladder (unpublished observations). Recently, we found that thaliporphine increased rhythmic activity when applied cumulatively to guinea-pig ileum. In this study, we evaluated the mechanism of action of thaliporphine-induced contraction in the guinea-pig ileum.
CaC12 1.9, K H 2 P O 4 1.2, N a H C O 3 25 and glucose 11.7 mM. The Krebs solution was aerated continuously with a gas mixture of 95% 0 2 and 5% CO 2. Ileal segments (2 cm long) were mounted in an organ bath (5 ml) containing Krebs solution at 37°C. The muscle strips were initially under a resting tension of 1 g. After an equilibration period of 60 min the strips were challenged with the test drugs. Changes in tension were recorded isometrically with a Grass FT03 force-displacement transducer connected to a Grass polygraph. When the effects of antagonists were studied, the muscle strip was pretreated with each antagonist for 15 min before thaliporphine was added. Results are expressed as means + S.E.M. of tension changes (in g) for n independent experiments. Student's t-test was used to assess the significant of differences between means. A P value of < 0.05 was considered as a significant difference.
2. Materials and methods
2.1. Isolated guinea-pig ileal preparations Guinea-pigs of either sex weighing 300-400 g were killed by a blow to the head and were bled. Segments of the terminal ileum, from 2 cm of the ileo-caecal valve, were removed quickly and placed in Krebs solution composed of NaC1 118.3, KC1 4.7, MgSO 4 1.2,
Correspondence to: C.M. Teng, Pharmacological Institute, College of Medicine, National Taiwan University, No. 1, Jen-Ai Road, Section l, Taipei, Taiwan.
2.2. Drugs Thaliporphine was prepared from isoboldine, isolated from plant Neolitsea konishii K (Lee and Yang, 1992), and its purity ( > 99%) was confirmed by N M R (nuclear magnetic resonance), mass, I R (infrared) and U V (ultraviolet) spectrum. The following drugs were used: tetrodotoxin, prazosin, phentolamine, propranolol, atropine, d i p h e n h y d r a m i n e , naloxone, indomethacin, nifedipine, staurosporine and verapamil. These drugs were obtained from Sigma Chemical CO. Bay K 8644 (methyl-l,4-dihydro-2,6-dimethyl-3-nitro-4(2-trifluoromet hylphenyl)-pyridine-5-carboxylat e) was
122 TABLE 1 Influence of various pretreatments on thaliporphine (10 ~M)-induced contractions in guinea-pig ileum. Treatment
Tension (g)
Control Tetrodotoxin (1 /x M) Phentolamine (10 txM) Prazosin (0.1 /x M) Propranolol (10 tiM) Naloxone (10 p.M) Atropine (0.1 ttM) Diphenhydramine (1 t~M) Methysergide (1 /xM) Indomethacin (10 # M ) Staurosporine (1 nM) Nifedipine (0.1 /xM) Verapamil (0.3/xM)
0,75 _+0.08 0,65 4- 0.11 0.704-0.10 0.80 _+0.09 0.69+0.08 0.65_+0.07 0.624-0.06 0.78 4- 0.07 0.75 4- 0.06 0.80 4- 0.10 0.70 + 0.05 0.00 + 0.00 0.00 _+0.08
1). The concentrations of these antagonists were sufficient to block the activity of the respective agonists (data not shown). Incubation of ileum with Ca2+-free Krebs solution (containing 1 mM E G T A ) for 15 min completely abolished the thaliporphine-induced contraction. However, addition of 3 mM CaC12 at the end of the experiment restored the thaliporphine-induced contraction to the control level. Pretreatment of ileum with nifedipine (0.03 /xM) and verapamil (0.03 /xM) shifted the concentration-response curve for thaliporphine to the right. Bay K 8644 (0.1 /xM) or KCI (20 mM), added 15 min before thaliporphine was added, had no effect on the resting tension but shifted the concentration response curve for thaliporphine to the left (fig. 1).
~' P < 0.001 as compared with the control.
4. Discussion
obtained from Bayer. When drugs were dissolved in dimethylsulfoxide (DMSO), the final concentration of DMSO in the bath did not exceed 0.1% (v/v) and had no effect on the muscle contraction.
3. Results
Cumulative application of 0.1-100 /xM thaliporphine produced a concentration-dependent contraction in the guinea-pig ileum. The geometric mean of the EDs0 was 3.6 + 0.7 /xM. The contractile response of the guinea-pig ileum to thaliporphine (10/xM) was not significantly modified in the presence of tetrodotoxin (1 /xM), phentolamine (10 /xM), prazosin (0.1 /xM), propranolol (10 /xM), naloxone (10 /xM), atropine (0.1 /xM), diphenhydramine (1 /xM), methysergide (1 /xM), indomethacin (10 /xM) or staurosporine (1 nM) (table
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Thaliporphine (p.M) Fig. i. Effects of nifedipine (0.03 ~ M , o), verapamil (0.03 izM, A), Bay K 8644 (0.I ~,M, A) and KCI (20 m M , Q) on the contraction induced by cumulative application of thaliporphine in guinea-pig ileum; (o) control. Agents to be tested were added 15 rain before thaliporphine was added. Each point represents the mean + S.E.M.
(n = 6-8).
The present results demonstrate that, in the guineapig ileum, thaliporphine caused a contractile response which was not affected by tetrodotoxin. This indicates that thaliporphine exerts a direct effect on intestinal smooth muscle and that activation of sodium channels is not essential for thaliporphine to induce contraction. However, Fontaine and Lebrun (1988) found that Bay K 8644 exerted potent spasmogenic effects on the isolated mouse distal colon. The presence of tetrodotoxin lowered the threshold concentration of Bay K 8644 and enhanced the maximal responses elicited by Bay K 8644. This finding suggests that the toxin interferes with inhibitory nerves, causing constant hyperpolarization of colonic smooth muscle cells. Tetrodotoxin, by reducing the hyperpolarization mediated by the neural influence, would therefore enhance the ability of Bay K 8644 to affect voltage-sensitive Ca 2+ channels. Moreover, dihydropyridine Ca 2+ channel binding sites are rather specific for Bay K 8644 and nifedipine, and neither verapamil nor diltiazem interfere competitively with the effect of Bay K 8644 (Schramm et al., 1983). However, both nifedipine and verapamil competitively antagonized the contraction induced by thaliporphine. This indicates that different binding sites for thaliporphine and Bay K 8644 may exist on the Ca 2+ channel. It is established that the tone of ileal smooth muscle is controlled by a dense innervation and many enteric neurotransmitters. Activation of ~- or/3-adrenoceptors (Broadley and Grassby, 1985) and opiate receptors reduces the tone. In contrast, activation of acetylcholine, serotonin or histamine receptors induces contraction. In the present study, the contractile effects of thaliporphine were not affected by antagonism of aadrenoceptors (phentolamine, prazosin), /3-adrenoceptors (propranolol), opiate (naloxone), muscarinic (atropine), serotonin (methysergide) or histamine (diphen-
123
hydramine) receptors. Thus, the thaliporphine-induced response is not mediated by receptors for the wellknown contractile or relaxing neurotransmitters. Some evidence suggests that protein kinase C, which is inhibited by staurosporine (Kiyoto et al., 1987; Schachtele et al., 1988), might regulate the tonic component of receptor-mediated vascular (Abdel-Latif, 1986) and intestinal smooth muscle contractions (Holzer and Lippe, 1989). The contractile effects of thaliporphine were not affected by staurosporine, thus ruling out the involvement of protein kinase C in the thaliporphine-induced contraction. In the rat aorta, the thaliporhine-induced contraction has been shown to depend on Ca 2+ influx through voltage-dependent Ca 2+ channels. In intestinal preparations, the thaliporphine-induced contraction was inhibited by nifedipine or verapamil and potentiated by Bay K 8644 or KC1. These results suggest that the influx of extracellular Ca 2 + via voltage-dependent Ca 2 + channels is mainly responsible for the intestinal contraction. However, the underlying mechanism for direct or indirect activation of voltage-dependent Ca 2+ channels remains to be clarified.
Acknowledgements This work was supported by a Research Grant of the National Science Council of the Republic of China (NSC81-0412-B002-113).
References Abdel-Latif, A.A., 1986, Calcium-mobilizing receptors, polyphosphoinositides, and the generation of second messengers, Pharmacol. Rev. 38, 227. Ahmad, R., J.M. Saa and M.P. Cava, 1977, Regioselective O-demethylation in the aporphine alkaloid series, J. Org. Chem. 42, 1228. Broadley, K.J. and P.F. Grassby, 1985, Alpha- and beta-adrenoceptor-mediated responses of the guinea-pig ileum and the effects of neuronal uptake inhibition, Naunyn-Schmiedeb. Arch. Pharmacol. 331,316. Fontaine, J. and P. Lebrun, 1988, Pharmacological analysis of the effects of Bay K 8644 and organic calcium antagonists on the mouse isolated distal colon, Br. J. Pharmacol. 94, 1198. Holzer, P. and I.T. Lippe, 1989, Protein kinase C may regulate the tonic component of intestinal smooth muscle contraction in res p o n s e to substance P, Naunyn-Schmiedeb. Arch. Pharmacol. 339, 314. Kiyoto, I., S. Yamamoto, E. Aizu and R. Kato, 1987, Staurosporine, a protein kinase C inhibitor, falls to inhibit 12-O-tetradecanoylphorbol-13-acetate-caused ornithine decarboxylase induction in isolated mouse epidermal cells, Biochem. Biophys. Res. Commun. 148, 740. Lee, S.S. and H.C. Yang, 1992, Isoquinoline alkaloids from Neolitsea konishii K, J. Chin. Chem. Soc. 39, 189. Schachtele, C.R., R. Seifert and H. Osswald, 1988, Stimulus-dependent inhibition of platelet aggregation by the protein kinase C inhibitors polymyxin B, H-7 and staurosporine, Biochem. Biophys. Res. Commun. 151,542. Schramm, M., G. Thomas, R. Towart and G. Franckowiak, 1983, Novel dihydropyridines with positive inotropic action through activation of Ca 2+ channels, Nature 303, 535.