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Effects of piperine on calcitonin gene-related peptide (CGRP)-containing nerves in the isolated rat atria
222
Neuroscience Letter.s. 91 i 1988) 222 22. Elsevier Scientific Publishers Ireland Ltd
NSL 05505
Effects of piperine on calcitonin gene-related peptide (CGRP)-containing nerves in the isolated rat atria Takashi Miyauchi l, Tomohisa Ishikawa 2, Yasuro Sagishita l, Akira Saito 2 and Katsutoshi Goto 2 1Department of Internal Medicine, Institute of Clinical Medicine and2Department of Pharmacology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba (Japan)
(Received 18 April 1988; Accepted 28 April 1988) Key words." Piperine; Calcitonin gene-related peptide; Non-adrenergic/non-cholinergic nerve; Heart; Rat
Piperine, a major pungent agent of black peppers, induced both positive chronotropic and inotropic responses in the isolated atria of rats. These responses were not blocked by various antagonists but exhibited rapid tachyphylaxis. After exposure to piperine in vitro, endogenous calcitonin gene-related peptide (CGRP) was profoundly depleted from intracardiac nerves. These results suggest that piperine releases endogenous CGRP from intracardiac non-adrenergic non-cholinergic nerves and that released CGRP exerts positive chronotropic and inotropic effects.
In addition to sympathetic adrenergic and p a r a s y m p a t h e t i c cholinergic nerves, cardiac function is controlled by non-adrenergic non-cholinergic ( N A N C ) nerves [1, 5, 1 1, 13, 15-17]. Pharmacological studies in vitro have shown that N A N C nerves in the heart produce positive c h r o n o t r o p i c and inotropic responses [5, 11, 15-17]. A m o n g cardioactive substances shown to be present in intracardiac nerves, calcitonin gene-related peptide ( C G R P ) , a 37-amino acid peptide [4], exerts both potent positive c h r o n o t r o p i c and inotropic effects and is likely to function as a neurotransmitter of N A N C nerves [11, 15, 17]. It has been d e m o n s t r a t e d that capsaicin, a pungent agent of red peppers, releases endogenous C G R P from intracardiac N A N C nerves and exerts both positive c h r o n o t r o p i c and inotropic effects in the isolated heart o f guinea pigs [3, 4, 11]. The cardiac effects o f capsaicin develop tachyphylaxis rapidly [3, 10, 1 1] because o f the depletion o f endogenous C G R P [11]. Pretreatment o f animals with capsaicin eliminated C G R P - I i k e immunoreactive ( C G R P - I ) nerves in the heart [15, 17], in which the cardiac effects o f capsaicin were also extinguished [3, 10]. Piperine, N-5-(3,4-methylenedioxyphenyl)penta-2,4-t,t-dienoyl piperidine, is a maj o r pungent agent of black peppers [19], and has an effect o f oral irritation like that
Correspondence." K. Goto, Department of Pharmacology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan.
0304-3940/88/$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.
223
of capsaicin [8]. However, the effects of piperine on the heart as well as on CGRP-I nerves are not known. In the present study, the effects of piperine on the rat atria were examined pharmacologically and immunohistochemically, to clarify the possible action of piperine on CGRP-I nerves in the heart. Male Wistar rats (250-300 g) were anesthetized with sodium pentobarbital (50 mg/ kg, i.p.). The hearts were quickly removed and placed into an ice-cold Krebs Ringer's solution of the following composition (mM):NaCI 113, KC1 4,8, CaCI2 2.2, KHzPO4 25, MgSO4 1.2, NaHCO3 25, and glucose 5.5. For the pharmacological studies in vitro, the right and left atria were individually set up in an organ bath containing 30 ml of the Krebs-Ringer's solution aerated with a mixture of 95% 02 5% CO2 and maintained at 37°C. Isometric contraction of both atrial strips was measured by a force displacement transducer (Nihon Kohden WT61 l, Tokyo, Japan). A resting tension of 1.0g was applied to the atria and at least 2 h were allowed for equilibration. The spontaneous beating rate of the right atrium was monitored by a tachograph (Nihon Kohden AT-600G). The left atrium was driven electrically at 3.3 Hz through a pair of platinum electrodes placed along both sides of the preparation at a distance of 5 mm. Square-wave pulses of 1 ms in duration and of a threshold intensity sufficient to elicit contractions (generally
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Fig. 1. A: the dose response curves of piperine in the right atria. Ordinate: peak increase in the heart rate. Abscissa: anti-log molar concentration of piperine. Because the effect of piperine developed rapid tachyphylaxis, the first response obtained in each tissue was utilized for constructing the dose response curves, i.e. only one response to a certain concentration of piperine in one tissue was used (n = 6). B: the tachyphylaxis to piperine. The left column represents the peak response to the first application of piperine (3 x 10 5 M) (n = 6), the right column represents the response to the second application of piperine (3 x 10 5 M) I h after the end of the first incubation with piperine (3 x 10 s M) for 15 min (n = 6).
224
a r o u n d l V, 8 m A ) were g e n e r a t e d by a N i h o n K o h d e n SEN-701 stimulator. C o n t r a c t i o n s were r e c o r d e d on a t h e r m a l p e n r e c o r d e r ( N i h o n K o h d e n W T - 6 8 7 G ) . F o r the i m m u n o h i s t o c h e m i c a l studies, the s p o n t a n e o u s l y b e a t i n g right atria and electrically driven left a t r i a (3.3 Hz) were i n c u b a t e d in a K r e b s - R i n g e r ' s s o l u t i o n with piperine (3 x 10 -5 M ) for 15 min a n d then w a s h e d with a fresh K r e b s - R i n g e r ' s solution for 1 h. R i g h t a n d left a t r i a i n c u b a t e d in a K r e b s - R i n g e r ' s solution without piperine served as controls. T h e tissues were then fixed with picric acid a n d p a r a f b r m a l d e h y d e , d e h y d r a t e d with e t h a n o l , cleared with xylene a n d r e h y d r a t e d as described p r e v i o u s l y [15, 17]. A n t i b o d i e s raised against rat C G R P ( C R B , C a m b r i d g e , U . K , ) were a p p l i e d at a dilution o f 1:1600 for 16 h at 4°C. The tissues were treated with b i o t i n y l a t e d a n t i - r a b b i t i m m u n o g l o b u l i n G (Vector Labs, Burlington, C A ) followed by fluorescein i s o t h i o c y a n a t e - l a b e l e d avidin (Vector Labs) m o u n t e d in buffered glycerol a n d e x a m i n e d u n d e r a Zeiss fluorescence m i c r o s c o p e . A p a i r e d t-test was used for the statistical analysis a n d a P value o f less t h a n 0.05 was a c c e p t e d as the significance o f the difference. Piperine caused a positive c h r o n o t r o p i c response o f the s p o n t a n e o u s l y b e a t i n g right a t r i a in a d o s e - d e p e n d e n t m a n n e r (Fig. I A). A f t e r a p p l y i n g piperine (3 x 10-5 M) for 15 min a n d a s u b s e q u e n t w a s h i n g for 1 h, p i p e r i n e (3 x 10 -5 M) was again
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Fig. 2. A: the dose--response curves of piperine in the left atria. Ordinate: peak increase in the tension. Abscissa: anti-log molar concentration of piperine. Because the effect of piperine again developed rapid tachyphylaxis, the first response obtained in each tissue was utilized for constructing the dose-response curves, i.e. only one response to a certain concentration of piperine in one tissue was used (n = 6). B: the tachyphylaxis to piperine. The left column represents the response to the first application of piperine (3 x 10-5 M) (n = 6): the right column represents the response to the second application of piperine (3 x 10 5 M) 1h after the end of the first incubation with piperine (3 x 10-5 M) for 15 rain (n = 6).
225 applied. The positive chronotropic response induced by the second application of piperine was greatly reduced compared to the first response (Fig. 1B), indicating that the effect of piperine on the right atria developed tachyphylaxis rapidly. The positive chronotropic response to piperine (3 × 10 5 M) was not affected significantly by atenolol (10 /~M) (fll-adrenoceptor antagonist), ICI-118,551 (1 /IM) (fl2-adrenoceptor antagonist), bunazosin (3/zM) (~l-adrenoceptor antagonist), atropine (1 /zM) (muscarinic antagonist), hexamethonium (3/tM) (nicotinic antagonist), diphenhydramine (1 /~M) (histamine Hwantagonist), cimetidine (3 tiM) (histamine Hz-antagonist) and methysergide (1/~M) (serotonin antagonist) (data not shown). Piperine also caused a positive inotropic response of the electrically driven left atria of rats in a dose-dependent manner (Fig. 2A). When piperine (3 x 10 -5 M) was applied for 15 rain and then washed out for I h, the inotropic response to piperine (3 × 10 5 M) was markedly reduced (Fig. 2B). Thus, the effect of piperine on the left atria again developed rapid tachyphylaxis. The positive inotropic response to piperine (3 x 10 5 M) was not affected significantly by the simultaneous presence ofatenolol (10/zM), ICI-118,551 (1 ¢tM), bunazosin (3/tM), atropine (1 /tM), hexamethonium (3/~M), diphenhydramine ( ! / t M ) , cimetidine (3/~M) and methysergide (1 /~M) (data not shown).
O Fig. 3. Calcitoningene-relatedpeptide (CGRP)-likeimmunoreactivenerves in the atria of rats. CGRP-like immunoreactive ncrves were found in the left atrium (A), The intensity of CGRP-like immunorcacfivity reduced greatly 1 h after the incubation with pipcrine(3 × 10 ~M) for 15 rain. (B). Bar- t00 jma.
226 CGRP-Iike immunoreactive nerves were found to be present in both the left (Fig. 3A) and right atria (not shown). One h after the incubation with piperine (3 x 10 -5 M, for 15 rain), the intensity of CGRP-like immunoreactivity was profoundly reduced in both the left (Fig. 3B) and right atria (not shown). The piperine-induced positive inotropic and chronotropic responses were not antagonized by fll-adrenergic, fl2-adrenergic, ~l-adrenergic, Hl-histaminergic, H2-histaminergic, serotoninergic, and nicotinic cholinergic antagonists. It has been shown that piperine reduces the level of substance P in the rat spinal cord [7, 12]. However, substance P and other tachykinins do not affect the heart rate or contraction of the isolated atria [10, 15, 17]. These findings indicate that the piperine-induced positive chronotropic and inotropic responses are mediated by neither biogenic amines nor tachykinins. C G R P has been shown to be present in intracardiac nerves and to exert both positive chronotropic and inotropic actions on the isolated atria of rats and guinea pigs [6, 15, 17]. The effects of piperine appear to be similar to those of capsaicin, e.g. both capsaicin-induced [3, 10] and piperine-induced cardiac effects were not blocked by various antagonists, exhibited rapid tachyphylaxis and were abolished when endogenous C G R P was depleted. Furthermore, endogenous C G R P was similarly depleted by in vitro application of capsaicin [2, 9, 1 1, 18]. Taken together, the results of the present study suggest that piperine, like capsaicin, releases and depletes C G R P from intracardiac N A N C nerves, and that released C G R P causes positive chronotropic and inotropic responses. Piperine itself appears to have no direct action on the atria. The depletion of C G R P may account for the lack of the cardiac effect of the second application of piperine. We thank Lisa G. Bond for advice in preparation of the manuscript. The present study was supported by the University of Tsukuba Project Research, the Research Foundation for Pharmaceutical Sciences and a Grant-in Aid from the Ministry of Education, Science and Culture of Japan. 1 Alter, W.A., III, Weiss, G.K. and Priola, D.V., Vagallyinduced tachycardia in atropinized dogs: effects offl-adrenergic blockade, Eur. J. Pharmacol., 24 (1973) 329-333. 2 Franco-Cereceda,A., Henke, H., Lundberg, J.M., Petermann, J.B., H6kfelt, ~. and Fischer, J.A., Calcitonin gene-related peptide (CGRP) in capsaicin-sensitivesubstance P-immunoreactivesensory neurons in animals and man: distribution and release by capsaicin, Peptides, 8 (1987) 399-410. 3 Franco-Cereceda, A. and Lundberg, J.A., Calcitonin gene-related peptide (CGRP) and capsaicininduced stimulation of heart contractile rate and force, Naunyn Schmiedeberg'sArch. Pharmacol., 331 (1985) 146-151. 4 Franco-Cereceda, A., Lundberg, J.A., Saria, A., Schreibmayer, W. and Tritthart, H.A., Calcitonin gene-related peptide: released by capsaicin and prolongation of the action potential in the guinea-pig heart, Acta Physiol. Scand., 132 (1988) 181 190. 5 Goto, K., Ishikawa, T., Kimura, S. and Saito, A., Intramural nerve-mediated inotropic responses of left atria of rats and guinea pigs: demonstration of alpha adrenergic and nonadrenergic noncholinergic responses in guinea pigs, J. Pharmacol. Exp. Ther., 243 (1987) 723-730. 6 Ishikawa, T., Okamura, N., Saito, A. and Goto, K., Effectsof calcitonin gene-related peptide (CGRP) and isoproterenol on the contractility and adenylate cyclase activity in the rat heart, J. Mol. Ceil. Cardiol., 19 (1987) 723 727.
227 7 Jhamandas, K., Yaksh, T.L., Harty, G., Szolcsanyi, J. and Go, V.L., Action of intrathecal capsaicin and its structural analogues on the content and release of spinal substance P: selectivity of action and relationship to analgesia, Brain Res., 23 (1984) 215 225. 8 Lawless, H. and Stevens, D.A., Effects of oral chemical irritation on taste, Physiol. Behav., 32 (1984) 995 998. 9 Lundberg, J.M., Franco-Cereceda, A., Hua, X., H6kfelt, T. and Fisher, J.A., Coexistence of substance P and calcitonin gene-related peptide-like immunoreactivities in sensory nerves in relation to cardiovascular and bronchoconstrictor effects of capsaicin, Eur. J. Pharmacol., 108 (1985) 315 319. I0 Lundberg, J.A., Hua, Y. and Fredholm, B.B., Capsaicin-induced stimulation of the guinea-pig atrium, Naunyn Smiedeberg's Arch. Pharmacol., 325 (1984) 176 182. I 1 Miyauchi, T., lshikawa, T., Sugishita, Y., Saito, A. and Goto, K., Effects of capsaicin on nonadrenergic noncholinergic nerves in the guinea pig atria: role of calcitonin gene-related peptide as cardiac ncurotransmitter, J. Cardiovasc. Pharmacol., 10 (1987) 675 682. 12 Micevych, P.E., Yaksh, T.L. and Szolcsanyi, J., Effects of intrathecal capsaicon analogues on the immunofluorescence of peptides and serotonin in the dorsal horn in rats, Neuroscience, 8 (1983) 123 131. 13 Rigel, D.F., Lipson, D. and Katona, P.G., Excess tachycardia: heart rate after antimuscarinic agents in conscious dogs, Am. J. Physiol., 246 (Heart Circ. Physiol., 15) (1984) H168 173. 14 Rosenfelt, M.G., Mermod, J.J., Amara, S.G., Swanson, L.W., Sawchenko, P., Rivier, J., Wale, W. and Evans, R., Production of a novel neuropeptide by the calcitonin gene via tissue specific RNA processing, Nature (Lond.), 304 (1983) 129 132. 15 Saito, A., Ishikawa, K., Kimura, S. and Goto, K., Role of calcitonin gene-related peptide as cardiotonic neurotransmitter in guinea pig left atria, J. Pharmacol. Exp. Ther., 243 (1987) 731 736. 16 Saito, A., Ishikawa, T., Masaki, T., Kimura, S. and Goto, K., Pharmacological analysis of autonomic innervation of the right atrium of rats and guinea pigs: demonstration of noradrenergic noncholinergic nerves, J. Pharmacol. Exp. Ther., 238 (1986) 713 719. 17 Saito, A., Kimura, S. and Goto, K., Calcitonin gene-related peptide as potential neurotransmitter in guinea pig right atrium, Am. J. Physiol., 250 (Heart Circ, Physiol., 19) (1986) H693 698. 18 Skofitsch, G. and Jacobowitz, D.M., Calcitonin gene-related peptide coexists with substance P in capsaicin sensitive neurons and sensory ganglia of the rat, Peptides, 6 (1985) 747 754. 19 Szolcsanyi, J., Capsaicin type pungent agents producing pyrexia. In A.S. Milton (Ed.), Handbook of Experimental Pharmacology: Pyretics and Antipyretics, Vol. 60, Springer, Berlin, 1982, pp. 437 478.
Neuroscience Letter.s. 91 i 1988) 222 22. Elsevier Scientific Publishers Ireland Ltd
NSL 05505
Effects of piperine on calcitonin gene-related peptide (CGRP)-containing nerves in the isolated rat atria Takashi Miyauchi l, Tomohisa Ishikawa 2, Yasuro Sagishita l, Akira Saito 2 and Katsutoshi Goto 2 1Department of Internal Medicine, Institute of Clinical Medicine and2Department of Pharmacology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba (Japan)
(Received 18 April 1988; Accepted 28 April 1988) Key words." Piperine; Calcitonin gene-related peptide; Non-adrenergic/non-cholinergic nerve; Heart; Rat
Piperine, a major pungent agent of black peppers, induced both positive chronotropic and inotropic responses in the isolated atria of rats. These responses were not blocked by various antagonists but exhibited rapid tachyphylaxis. After exposure to piperine in vitro, endogenous calcitonin gene-related peptide (CGRP) was profoundly depleted from intracardiac nerves. These results suggest that piperine releases endogenous CGRP from intracardiac non-adrenergic non-cholinergic nerves and that released CGRP exerts positive chronotropic and inotropic effects.
In addition to sympathetic adrenergic and p a r a s y m p a t h e t i c cholinergic nerves, cardiac function is controlled by non-adrenergic non-cholinergic ( N A N C ) nerves [1, 5, 1 1, 13, 15-17]. Pharmacological studies in vitro have shown that N A N C nerves in the heart produce positive c h r o n o t r o p i c and inotropic responses [5, 11, 15-17]. A m o n g cardioactive substances shown to be present in intracardiac nerves, calcitonin gene-related peptide ( C G R P ) , a 37-amino acid peptide [4], exerts both potent positive c h r o n o t r o p i c and inotropic effects and is likely to function as a neurotransmitter of N A N C nerves [11, 15, 17]. It has been d e m o n s t r a t e d that capsaicin, a pungent agent of red peppers, releases endogenous C G R P from intracardiac N A N C nerves and exerts both positive c h r o n o t r o p i c and inotropic effects in the isolated heart o f guinea pigs [3, 4, 11]. The cardiac effects o f capsaicin develop tachyphylaxis rapidly [3, 10, 1 1] because o f the depletion o f endogenous C G R P [11]. Pretreatment o f animals with capsaicin eliminated C G R P - I i k e immunoreactive ( C G R P - I ) nerves in the heart [15, 17], in which the cardiac effects o f capsaicin were also extinguished [3, 10]. Piperine, N-5-(3,4-methylenedioxyphenyl)penta-2,4-t,t-dienoyl piperidine, is a maj o r pungent agent of black peppers [19], and has an effect o f oral irritation like that
Correspondence." K. Goto, Department of Pharmacology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan.
0304-3940/88/$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.
223
of capsaicin [8]. However, the effects of piperine on the heart as well as on CGRP-I nerves are not known. In the present study, the effects of piperine on the rat atria were examined pharmacologically and immunohistochemically, to clarify the possible action of piperine on CGRP-I nerves in the heart. Male Wistar rats (250-300 g) were anesthetized with sodium pentobarbital (50 mg/ kg, i.p.). The hearts were quickly removed and placed into an ice-cold Krebs Ringer's solution of the following composition (mM):NaCI 113, KC1 4,8, CaCI2 2.2, KHzPO4 25, MgSO4 1.2, NaHCO3 25, and glucose 5.5. For the pharmacological studies in vitro, the right and left atria were individually set up in an organ bath containing 30 ml of the Krebs-Ringer's solution aerated with a mixture of 95% 02 5% CO2 and maintained at 37°C. Isometric contraction of both atrial strips was measured by a force displacement transducer (Nihon Kohden WT61 l, Tokyo, Japan). A resting tension of 1.0g was applied to the atria and at least 2 h were allowed for equilibration. The spontaneous beating rate of the right atrium was monitored by a tachograph (Nihon Kohden AT-600G). The left atrium was driven electrically at 3.3 Hz through a pair of platinum electrodes placed along both sides of the preparation at a distance of 5 mm. Square-wave pulses of 1 ms in duration and of a threshold intensity sufficient to elicit contractions (generally
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Fig. 1. A: the dose response curves of piperine in the right atria. Ordinate: peak increase in the heart rate. Abscissa: anti-log molar concentration of piperine. Because the effect of piperine developed rapid tachyphylaxis, the first response obtained in each tissue was utilized for constructing the dose response curves, i.e. only one response to a certain concentration of piperine in one tissue was used (n = 6). B: the tachyphylaxis to piperine. The left column represents the peak response to the first application of piperine (3 x 10 5 M) (n = 6), the right column represents the response to the second application of piperine (3 x 10 5 M) I h after the end of the first incubation with piperine (3 x 10 s M) for 15 min (n = 6).
224
a r o u n d l V, 8 m A ) were g e n e r a t e d by a N i h o n K o h d e n SEN-701 stimulator. C o n t r a c t i o n s were r e c o r d e d on a t h e r m a l p e n r e c o r d e r ( N i h o n K o h d e n W T - 6 8 7 G ) . F o r the i m m u n o h i s t o c h e m i c a l studies, the s p o n t a n e o u s l y b e a t i n g right atria and electrically driven left a t r i a (3.3 Hz) were i n c u b a t e d in a K r e b s - R i n g e r ' s s o l u t i o n with piperine (3 x 10 -5 M ) for 15 min a n d then w a s h e d with a fresh K r e b s - R i n g e r ' s solution for 1 h. R i g h t a n d left a t r i a i n c u b a t e d in a K r e b s - R i n g e r ' s solution without piperine served as controls. T h e tissues were then fixed with picric acid a n d p a r a f b r m a l d e h y d e , d e h y d r a t e d with e t h a n o l , cleared with xylene a n d r e h y d r a t e d as described p r e v i o u s l y [15, 17]. A n t i b o d i e s raised against rat C G R P ( C R B , C a m b r i d g e , U . K , ) were a p p l i e d at a dilution o f 1:1600 for 16 h at 4°C. The tissues were treated with b i o t i n y l a t e d a n t i - r a b b i t i m m u n o g l o b u l i n G (Vector Labs, Burlington, C A ) followed by fluorescein i s o t h i o c y a n a t e - l a b e l e d avidin (Vector Labs) m o u n t e d in buffered glycerol a n d e x a m i n e d u n d e r a Zeiss fluorescence m i c r o s c o p e . A p a i r e d t-test was used for the statistical analysis a n d a P value o f less t h a n 0.05 was a c c e p t e d as the significance o f the difference. Piperine caused a positive c h r o n o t r o p i c response o f the s p o n t a n e o u s l y b e a t i n g right a t r i a in a d o s e - d e p e n d e n t m a n n e r (Fig. I A). A f t e r a p p l y i n g piperine (3 x 10-5 M) for 15 min a n d a s u b s e q u e n t w a s h i n g for 1 h, p i p e r i n e (3 x 10 -5 M) was again
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Fig. 2. A: the dose--response curves of piperine in the left atria. Ordinate: peak increase in the tension. Abscissa: anti-log molar concentration of piperine. Because the effect of piperine again developed rapid tachyphylaxis, the first response obtained in each tissue was utilized for constructing the dose-response curves, i.e. only one response to a certain concentration of piperine in one tissue was used (n = 6). B: the tachyphylaxis to piperine. The left column represents the response to the first application of piperine (3 x 10-5 M) (n = 6): the right column represents the response to the second application of piperine (3 x 10 5 M) 1h after the end of the first incubation with piperine (3 x 10-5 M) for 15 rain (n = 6).
225 applied. The positive chronotropic response induced by the second application of piperine was greatly reduced compared to the first response (Fig. 1B), indicating that the effect of piperine on the right atria developed tachyphylaxis rapidly. The positive chronotropic response to piperine (3 × 10 5 M) was not affected significantly by atenolol (10 /~M) (fll-adrenoceptor antagonist), ICI-118,551 (1 /IM) (fl2-adrenoceptor antagonist), bunazosin (3/zM) (~l-adrenoceptor antagonist), atropine (1 /zM) (muscarinic antagonist), hexamethonium (3/tM) (nicotinic antagonist), diphenhydramine (1 /~M) (histamine Hwantagonist), cimetidine (3 tiM) (histamine Hz-antagonist) and methysergide (1/~M) (serotonin antagonist) (data not shown). Piperine also caused a positive inotropic response of the electrically driven left atria of rats in a dose-dependent manner (Fig. 2A). When piperine (3 x 10 -5 M) was applied for 15 rain and then washed out for I h, the inotropic response to piperine (3 × 10 5 M) was markedly reduced (Fig. 2B). Thus, the effect of piperine on the left atria again developed rapid tachyphylaxis. The positive inotropic response to piperine (3 x 10 5 M) was not affected significantly by the simultaneous presence ofatenolol (10/zM), ICI-118,551 (1 ¢tM), bunazosin (3/tM), atropine (1 /tM), hexamethonium (3/~M), diphenhydramine ( ! / t M ) , cimetidine (3/~M) and methysergide (1 /~M) (data not shown).
O Fig. 3. Calcitoningene-relatedpeptide (CGRP)-likeimmunoreactivenerves in the atria of rats. CGRP-like immunoreactive ncrves were found in the left atrium (A), The intensity of CGRP-like immunorcacfivity reduced greatly 1 h after the incubation with pipcrine(3 × 10 ~M) for 15 rain. (B). Bar- t00 jma.
226 CGRP-Iike immunoreactive nerves were found to be present in both the left (Fig. 3A) and right atria (not shown). One h after the incubation with piperine (3 x 10 -5 M, for 15 rain), the intensity of CGRP-like immunoreactivity was profoundly reduced in both the left (Fig. 3B) and right atria (not shown). The piperine-induced positive inotropic and chronotropic responses were not antagonized by fll-adrenergic, fl2-adrenergic, ~l-adrenergic, Hl-histaminergic, H2-histaminergic, serotoninergic, and nicotinic cholinergic antagonists. It has been shown that piperine reduces the level of substance P in the rat spinal cord [7, 12]. However, substance P and other tachykinins do not affect the heart rate or contraction of the isolated atria [10, 15, 17]. These findings indicate that the piperine-induced positive chronotropic and inotropic responses are mediated by neither biogenic amines nor tachykinins. C G R P has been shown to be present in intracardiac nerves and to exert both positive chronotropic and inotropic actions on the isolated atria of rats and guinea pigs [6, 15, 17]. The effects of piperine appear to be similar to those of capsaicin, e.g. both capsaicin-induced [3, 10] and piperine-induced cardiac effects were not blocked by various antagonists, exhibited rapid tachyphylaxis and were abolished when endogenous C G R P was depleted. Furthermore, endogenous C G R P was similarly depleted by in vitro application of capsaicin [2, 9, 1 1, 18]. Taken together, the results of the present study suggest that piperine, like capsaicin, releases and depletes C G R P from intracardiac N A N C nerves, and that released C G R P causes positive chronotropic and inotropic responses. Piperine itself appears to have no direct action on the atria. The depletion of C G R P may account for the lack of the cardiac effect of the second application of piperine. We thank Lisa G. Bond for advice in preparation of the manuscript. The present study was supported by the University of Tsukuba Project Research, the Research Foundation for Pharmaceutical Sciences and a Grant-in Aid from the Ministry of Education, Science and Culture of Japan. 1 Alter, W.A., III, Weiss, G.K. and Priola, D.V., Vagallyinduced tachycardia in atropinized dogs: effects offl-adrenergic blockade, Eur. J. Pharmacol., 24 (1973) 329-333. 2 Franco-Cereceda,A., Henke, H., Lundberg, J.M., Petermann, J.B., H6kfelt, ~. and Fischer, J.A., Calcitonin gene-related peptide (CGRP) in capsaicin-sensitivesubstance P-immunoreactivesensory neurons in animals and man: distribution and release by capsaicin, Peptides, 8 (1987) 399-410. 3 Franco-Cereceda, A. and Lundberg, J.A., Calcitonin gene-related peptide (CGRP) and capsaicininduced stimulation of heart contractile rate and force, Naunyn Schmiedeberg'sArch. Pharmacol., 331 (1985) 146-151. 4 Franco-Cereceda, A., Lundberg, J.A., Saria, A., Schreibmayer, W. and Tritthart, H.A., Calcitonin gene-related peptide: released by capsaicin and prolongation of the action potential in the guinea-pig heart, Acta Physiol. Scand., 132 (1988) 181 190. 5 Goto, K., Ishikawa, T., Kimura, S. and Saito, A., Intramural nerve-mediated inotropic responses of left atria of rats and guinea pigs: demonstration of alpha adrenergic and nonadrenergic noncholinergic responses in guinea pigs, J. Pharmacol. Exp. Ther., 243 (1987) 723-730. 6 Ishikawa, T., Okamura, N., Saito, A. and Goto, K., Effectsof calcitonin gene-related peptide (CGRP) and isoproterenol on the contractility and adenylate cyclase activity in the rat heart, J. Mol. Ceil. Cardiol., 19 (1987) 723 727.
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