Inhibition of contractile tension by capsaicin in isolated rat papillary muscle

ISSN 0306-3623/96 $15.00 + .00 SSDI 0306-3623(95)00093-3 All rights reserved

Gen. Pharmac. Vol. 27, No. 1, pp. 129-132, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA. ELSEVIER

Inhibition of Contractile Tension by Capsaicin in Isolated Rat Papillary Muscle T. Yamato, M. Aomine,* M. Ikeda, H. Noto and C. Ohta DIVISION OF NUTRITIONAL PHYSIOLOGY, THE GRADUATE SCHOOL OF HEALTH AND NUTRITIONAL SCIENCES, NAKAMURA GAKUEN UNIVERSITY, BEFU, FUKUOKA, JAPAN

A B S T R A C T . 1. We e x a m i n e d effects of capsaicin ( 1 0 - 9 - 1 0 -s M), a p u n g e n t agent extracted from red pepper, o n the contractile tension of rat ventricular papillary muscles stimulated at various cycle lengths (0.2, 0.5, 1, 2 a n d 5 sec). 2. Capsaicin produced a m a r k e d c o n c e n t r a t i o n - d e p e n d e n t decrease in the amplitude, the rate of rise (dp/dt) a n d the rate of relaxation (dr/d0 of the tension. 3. However, the half relaxation time a n d the time to peak t e n s i o n of the tension were slightly affected by the agent. 4. T h e negative inotropic effect of capsaicin was stimulus cycle length (CL) d e p e n d e n t . I n particular, ICs0 (50% inhibitive concentration) of the agent i n the amplitude of the tension was stimulus CL d e p e n d e n t . T h a t is, the values of ICs0 were a r o u n d 10 -~ M at longer CLs (1, 2 a n d 5 sec), a n d the value of ICs0 at CL 0.2 sec was 4 x 10 -6 M. 5. These capsaicin-induced negative inotropic effects were reversible. Other studies from our laboratory show that the negative inotropic effects may be largely due to a decrease i n Ca z + current. GEN PHARMAC 27;1:129-132, 1996. KEY W O R D S . Capsaicin, negative inotropic effect, rat cardiac muscle

INTRODUCTION Capsaicin (8-methyl-N-nanillyl-6-nonenamide), the pungent agent present in various species of hot peppers, Capsicum, has many physiological and pharmacological actions (Szolcsanyi and Jancso-Gabor, 1973; Szolcsanyi, 1982; Hori, 1984). The pharmacological study of this agent can be traced back more than a century when Hogyes (1878) found hypothermia in the dog after intragastric administration of an oily extract of paprika (Hungarian red pepper). Later, Jancso and his colleagues (1977) revealed a broad range of physiological actions of capsaicin on circulation, respiration, the gastrointestinal system, the sensory system and thermoregulation. Capsaicin has both direct and reflexogenic actions on the cardiovascular system (Virus and Gebhart, 1979). In the isolated atrium from guinea pig, capsaicin caused a positive inotropic and chronotropic effect, which was not mediated via catecholamines (Fukuda and Fujiwara, 1969; Molnar et al., 1969; Toda et al., 1972; Lundberg et al., 1984; Zernig et al., 1984). It is known that the capsaicin-sensitive substance P (SP) nerve is present in the guinea pig auricle (Papka et al., 1981; Murphy et al., 1982; Lundberg et al., 1983). Although Butcher et al. (1977) and Kulakowski et al. (1983) reported no clearcut stimulatory response of SP in the heart, the effects ofcapsaicin on the isolated guinea pig auricle may be due to SP release. However, the underlying mechanisms in its action are not yet clarified. In the present study, to further clarify the effects of capsaicin, we investigate the capsaicin effects on the contractile tension of the ventricular papillary muscles from rat, which is known to show a negative staircase phenomenon. That is, the amplitude of the contractile tension is generally smaller, when stimulus frequency is higher. To study the effects of different stimulus frequency on the capsaJ.cin effects, the muscle preparations were stimulated at five different stimulus frequencies (cycle *To whom all correspondence should be addressed• Received 2 February 1995.

length 0.2, 0.5, 1, 2 and 5 sec). Further, as it is reported that capsaicin produced a positive inotropic action at the low concentrations (Molnar et al., 1969; Toda et al., 1972; Zernig et al., 1984), we studied the effects of b igher concentrations as well as the low concentrations of the agent. MATERIALS AND METHODS Preparation, solution and measurement of isometric tension Wistar rats of either sex weighing 250-300 g were anesthetized by ethyl ether and killed by a blow on the neck. The hearts were rapidly removed and immersed in oxygenated Tyrode's solution (see below) at room temperature (20-22 ° C). The papillary muscles with a length of 0.5-1.0 mm and a diameter o f < 0.5 mm were excised from the right and left ventricles, mounted in a Lucite tissue chamber (1.5 ml) and superfused continuously with oxygenated Tyrode's solution at the flow rate of 3 ml/min. The tendinous end of the muscle was connected to a force transducer (Nihon Kohden TB-612T) via fine silk thread to record isometric tension. The muscle was stretched to the length at which the developed twitch tension reached maximum. The Tyrode's solution contained (in raM) 145 NaCI, 4 KCI, 1.8 CaCI2, 1.1 MgCI2, 5 HEPES, 10.9 glucose. The pH of the solution was adjusted at 7.4 by 1 N NaOH. The temperature was maintained at 36 _+ 0.5 °C. The preparations were equilibrated with control Tyrode's solution for > 1 hr before the start of the study. • \ . The following twitch tension characteristics were measured: amplitude of twitch tension (amplitude), time to peak tension (TPT), contracting speed (dp/dt), relaxing speed (dr/dr) and half relaxation time (T1/2) (Fig. 1). Drugs

Capsaicin was obtained from Wako Chemicals (Osaka, Japan). Capsaicin was diluted to the concentration from a 10 3 M stock solution.

T. Yamato et al.

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Statistical analysis Results are expressed as means_+SD. Data were analyzed using nonpaired Student's t-test, and P < 0.05 was considered statistically significant. RESULTS Figure 2 shows typical steady-state effects of 10 -9 and 10 5 M capsaicin on the twitch tension (7) and the dT/dt from rat papillary muscle stimulated at cycle length (CL) 5, 2, 1, 0.5 and 0.2 sec. Capsaicin (10 9 10-5 M) produced concentration- and stimulus frequency-dependent decreases in the amplitude, TPT, T~/2, dp/dt and dr/dt of the tension (Figs. 3-8). In particular, capsaicin depressed the amplitude, dp/dt and dr/dt of the tension in a concentration-dependentmanner (Figs. 3 and 4), compared with TPT and TI/2 (Figs. 5 and 6). For instance, the lowest concentration (10-9 M) used in the present study decreased the amplitude, dp/dt and dr/dt at CL 5 sec, by 32, 33 and 35%, respectively. However, the same concentration of the drug decreased the TPT and T~/2 only by 18 and 2%, respectively. Capsaicin I0 -5 M decreased the

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FIGURE 3. Effects of various concentrations (10-9-10 -s M) of capsaicin and different stimulus cycle length (CL 5-0.2 sec) on the amplitude of twitch tension of rat ventricular papillary muscles. Amplitude (ordinate) during application of capsaicin expressed, in each CL, as a percentage of the amplitude (100%) measured at CL 5 sec in the absence of capsaicin, as a function of CL (abscissae). *P < 0.05 and #P < 0.01, compared with controls measured at corresponding CL. amplitude, dp/dt and dr~dr by 73, 64 and 66% at CL 5 sec, respectively, but decreased the TPT and T1/2 only by 30 and 8%, respectively. To further characterize the effects of stimulus frequency on the action ofcapsaicin, the amplitude or TPT was plotted against the concentration of capsaicin at various stimulus cycle lengths (CL 5, 2, 1, 0.5 and 0.2 sec) (Figs. 7 and 8). The amplitude was chosen as a representative of

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FIGURE 2. A typical example of effects of capsaicin 10 -9 and 10 -s M on twitch tension from rat ventricular papillary muscle at CL 5-0.2 sec. The preparation was stimulated for _ 3 rain at each CL to ensure steady-state conditions. Top, twitch tension (T) and the differentiation (dT/dt) at CL 5-0.2 sec in control Tyrode's solution. Middle and bottom, effects of 10 -9 and 10-s M of capsaicin, respectively.

FIGURE 4. Effects of various concentrations (10-9-10 -s M) of capsaicin and different stimulus cycle length (CL 5-0.2 sec) on the dp/dt of twitch tension (A) and dr/dr of twitch tension (B) of rat ventricular papillary muscles. Both dp/dt and drldt during applica. tion of capsaicin expressed, in each CL, as a percentage of the dp/ dt (100%) or dr/dt (100%) measured at CL 5 sec in the absence of capsaicin. Other legends are the same as in Fig. 3.

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stimulus CLs. We found the following: (1) capsaicin produced a marked decrease in the amplitude, the dp/dt and the dr/dt of the tension in a concentration-dependent manner. (2) However, Ti/2 and TPT of the tension were slightly affected by the agent. (3) The negative inotropic effects of capsaicin were stimulus CL dependent. In particular, the IC50 of the agent in the amplitude of the tension depended on stimulus CL compared with ICs0 in the TPT. In the present study, we found a marked negative inotropic effect of capsaicin in the rat ventricular muscle. In atrial preparation, however, Molnar et al. (1969) found that capsaicin (5 x 10-s-10 -7 g/ml) exerted positive inotropic and chronotropic effects, which were not antagonized by propranolol or cocaine. Later, Zernig et al. (1984) reported that

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FIGURE 7. T h e concentration and stimulus CL dependency of capsaicin effects on amplitude of twitch tension of rat ventricular papillary muscles. T h e data of Fig. 3 are re-plotted in this figure, in which the value of amplitude at each concentration of capsaicin (ordinate) is relative to the value (100%) in the absence of capsaicin (control condition) as a function of capsaicin concentration (ab. scissae).

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concentration-dependent action of capsaicin, whereas the TPT was a representative of concentration non-dependence. At each CL, the amplitude or TPT w as represented relative to the value (100%) in control Tyrode's solution. As shown in Fig. 7, the amplitude of tension decreased with increasing both the concentration of capsaicin and stimulus CL. O n the other hand, a little depression of TPT was observed in the presence of various concentrations ofcapsaicin (Fig. 8). At low stimulus frequencies, a decrease by capsaicin in TPT was larger. So far as the amplitude of tension is concerned, as described before, the depression by capsaicin of amplitude was stimulus C L dependent (Fig. 7). At each CL, a half-maximal inhibition (IC~) of amplitude by capsaicin was calculated from the data of Fig. 7. An ICs0 obtained at each CL is plotted vs. CL (Fig. 9). As shown in Fig. 9, an IC50 at CL 0.2 sec was 4 x 10 -6 M. However, the value became smaller as CL increased. That is, IC50 varied with stimulus frequency.

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FIGURE 5. Effects of various concentrations ( 1 0 - 9 - 1 0 -s M) of capsaicin and different stimulus cycle length (CL 5-0.2 sec) on T P T of twitch tension of rat ventricular papillary muscles. T P T during application of capsaicin expressed, in each CL, as a percent. age of the T P T (100%) measured at CL 5 sec in the absence of capsaicin. Other legends are the same as in Fig. 3.

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FIGURE 6. Effects of various concentrations ( 1 0 - 9 - 1 0 -s M) of capsaicin and different stimulus cycle length (CL 5 - 0 . 2 sec) on Tlfz of twitch tension of rat ventricular muscles. Tl~zduring application of capsaicin expressed, in each CL, as a percentage of the T,/z (100%) measured at CL 5 sec in the absence of capsaicin. Other legends are the same as in Fig. 3.

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132

T. Yamato et al. hard to explain why there exists the differential effect of capsaicin in between atrium and ventricle. The most straightforward possibility is t h a t the density of [3-receptor in the atrial muscle may differ from that in the ventricular muscle. In conclusion, the negative inotropic effect ofcapsaicin may be largely due to its inhibitory action o n the Ca 2+ current. However, further investigation should be carried out to clarify the electrophysiological action of capsaicin o n heart muscle.

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The present study was supported in part by a Grant-in-Aid (No. 05680043) for General Scientific Research from the Ministry of Education, Science and Culture of Japan to M.A. and by grants (No. 9029 and No. 92044) from the Salt Science Research Foundation, Japan to M.A. 0

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Cycle Length (sec) F I G U R E 9. T h e r e l a t i o n s h i p b e t w e e n ICs0 o f c a p s a i c i n a n d stimulus CL. ICs0 (ordinate) is p l o t t e d vs. C L (abscissae). ICs0 is obtained f r o m the data i n Fig. 7.

capsaicin (0.03-3.3 x 10 -6 M) first increased and t h e n inhibited the contractile tension of the atrial muscle, whereas its effect o n the ventricular muscle was of a predominantly inhibitory nature, using guinea pig. T h e increase in contractions in the presence of capsaicin was associated with an inhibition of the upstroke velocity of the action potential in b o t h the atrial and papillary muscle preparations, without a prolongation of action potential duration. They concluded that the positive inotropic effect of capsaicin in the atrium may be due to an increase in [Ca2+]~, which does not result from a transmembraneous influx of Ca 2+ but probably from a release of C a 2+ from stores. T h e negative inotropic effect of capsaicin may be explained by an unspecific membranestabilizing effect ofcapsaicin. The C a 2+ released by c apsaicin contributes to the trigger Ca 2+ (for triggering a contraction a certain a m o u n t of C a 2÷) t h a t has to bind to the sarcoplasmic reticulum (SR) (for a review, see Fabiato a n d Fabiato, 1979). In short, after the Ca 2+ released by capsaicin has been extruded from the cell, the quinidine-like membranestabilizing effect of capsaicin becomes visible as a decrease in the size of contraction (Zernig et al., 1984). We previously demonstrated that capsaicin has a dual effect on the guinea pig ventricular papillary muscle: a positive inotropic effect at low concentration ( - 10 7 M ) o f capsaicin and a negative inotropic effect at higher concentrations (Aomine a n d Ehara, 1993). The positive inotropic effect of capsaicin was markedly attenuated in the presence of propranolol (10 s M), strongly suggesting the involvement of [3-receptor in the capsaicin-induced positive inotropic action. In ventricular myocytes, Ca 2+ current was profoundly decreased by capsaicin, in association with shortening of action potential duration (Aomine and Ehara, 1993). Therefore, the negative inotropic effect of capsaicin may be partly due to the decrease of Ca 2+ current. In addition, in an accompanying paper (Yamato et al., 1996), we showed that capsaicin may not inhibit a Ca 2+ release from SR in the rat ventricular papillary muscle. Zernig et al.'s (1984) observations with regard to the ventricular muscle of guinea pig are in good accord with our results, using the rat papillary muscle, a predominantly inhibitory effect of capsaicin. It is

References Aomine M. and Ehara T. (1993) Electrophysiological effects of capsaicin on the ventricular muscle and myocytes of guinea-pig heart. Jpn. J. Physiol. 43 (Suppl.2), s155. Burcher E., Atterhog J. H., Pernow B. and Rossell S. (1977) Cardiovascular effects of substance P: effects on the heart and religional blood flow in the dog. In: Substance P (Edited by yon Euler U. S. and Pernow B.), pp. 261-268. Raven Press, New York. Fabiato A. and Fabiato F. (1979) Calcium and cardiac excitation-contraction coupling. Annu. Rev. Physiol. 41, 473-484. Fukuda N. and Fujiwara M. (1969) Effect of capsaicin on the guinea-pig isolated atrium. J. Pharm. Pharmac. 21, 622-624. Hogyes A. (1878) Beitrage zur physiologischen Wirkung der Bestandteile des Capsicum annuum. Arch. exp. Path. Pharmak. 9, 117-130. Hod T. (1984) Capsaicin and central control of thermoregulation. Pharmac. Ther. 26, 389-416. Jancso G., Kiraly E. and Jancso-Gabor A. (1977) Pharmacologically induced selective degeneration of chemosensitive primary sensory neurons. Nature, Lond. 270, 741-743. Kulakowski E. C., Lampson W. G., Schaffer W. and Lovenberg W. (I983) Action of substance P on the working heart. Life Sci. 32, 1017-1100. Lundberg J. M., Brodin E. and Saria A. (1983) Effects and distribution of vagal capsaicin-sensitive substance P neurons with special reference to the trachea and lungs. Acta Physiol. Scand. 9, 243-252. LundbergJ. M., Hua Y. and Fredholm B. B. (1984) Capsaicin-induced stimulation of the guinea-pig atrium. Involvement of a novel transmitter or a direct action on myocytes? Naunyn-Schmiedeberg's Arch. Pharmac. 325, 176-182. Molnar J., Gyorgy L., Unyi G. and Kenyeres J. (1969) Effect of capsaicin on the isolated ileum and auricle of the guinea pig. Acta Physiol. Acad. Sci. Hung. 8, 341-349. Murphy R., Fu mess J. B., Beardsley A. M. and Costa M. (1982) Characterization of substance P-like immunoreactivity in peripheral sensory nerves and enteric nerves by high pressure liquid chromatography and radioimmunoassay. Regul. Rep. 4, 203-212. Papka R. E., Furness J. B., Della N. G. and Costa M. (1981) Depletion by capsaicin of substance P immunoreactivity and acetylcholinesterase activity from nerve fibres in the guinea-pig heart. Neurosci. Lett. 27, 47-53. Szolcsanyi J. (1982) Capsaicin type pungent agents producing pyrexia. In: Handbook of Experimental Pharmacology (Edited by Milton A. S.), Vol. 60, pp. 437478. Springer-Verlag, Berlin Heidelberg. Szolcsanyi J. and Jancso-Gabor A. (1973) Capsaicin and other pungent agents as pharmacological tools in studies on thermoregulation. In: The Pharmacology of Thermoregulation (Edited by Schonbaum E. and Lomax P.), pp. 395-409. Karger, Basel. Toda N., Usui H., Nishino N. and Fujiwara M. (1972) Cardiovascular effects of capsaicin in dogs and rabbits. J. Pharmac. exp. Ther. 181, 512-52I. Virus R. M. and Gebhart G.-F. (1979) Pharmacological actions of capsaicin: Apparent involvement of substance P and serotonin. Life Sci. 25, 1273-1284. Yamato T., Aomine M., Ikeda M., Noto H. and Ohta C. (1996) Capsaicin does not inhibit the intracellular calcium handling process in rat ventricular papillary muscle? Gen. Pharmac. (in press). Zernig G., Holzer P. and Lembeck F. (1984) A study of the mode and site of action of capsaicin in guinea-pig heart and rat uterus. Naunyn-Schmiedeberg's Arch. Pharmac. 326, 58-63.