European Journal of Pharmacology, 186 (1990) 71-77 Elsevier
71
EJP 51469
Effects of pipeline on the motility of the isolated guinea-pig ileum: comparison with capsaicin M i y a k o Takaki, J i - G u a n g Jill, Y u n - F e i L u a n d Sosogu N a k a y a r n a Department of Physiology, Okayama University Medical School, Shikatacho, Okayama 700, Japan Received 30 October 1989,revised MS received 29 May 1990, accepted 19 June 1990
Piperine (1 gM), a congener of capsaicin, produced an initial contraction, blocked the capsaicin-sensitive contractile response to mesenteric nerve stimulation and inhibited the twitch response induced by field stimulation in the isolated guinea-pig ileum. These three effects of piperine (1 /zM) were rapidly desensitized and significantly antagonized by ruthenium red (0.5-1 /zM), an inorganic dye known to antagonize the effects of capsaicin. The contractile effect of piperine was abolished by application of tetrodotoxin plus desensitization with substance P or by extrinsic denervation. The inhibitory effect of piperine (1/tM) on the twitch response was antagonized by desensitization with calcitonin gene-related peptide (CGRP). Moreover, cross-tachyphylaxis between piperine and capsaicin was observed, suggesting that a similar mechanism may be involved in the effects of these agents. The contractile effects induced by piperine (10/tM) and the subsequent inhibitory effects on the twitch response were not desensitized and largely persisted after extrinsic denervation. The contractile effects of piperine (10 pM) were not strongly inhibited by tetrodotoxin plus desensitization with substance P. It was concluded that the lower concentration of piperine caused contraction and inhibited the twitch responses by releasing substance P arid CGRP, respectively, from sensory nerves, and blocked the response to mesenteric nerve stimulation by a mechanism similar to that of capsaicin. At higher concentrations, piperine had non-specific direct actions on the smooth muscle. Piperine; Capsaicin; Calcitonin gene-related peptide (CGRP); Substance P; Ruthenium red; Ileal motility; (Guinea-pig)
1. Introduction
The stimulating effect of capsaicin, the pungent substance in red pepper, is specific for chemosensitive sensory nerves at low concentrations (1/~M) and there is no evidence that it has a direct effect on efferent neuronal structures (Toh et al., 1955; Jancs6 et al., 1967; Szolcsfinyi and Jancs6-G~bor, 1975; Barth6 and Szolcsfinyi, 1978), although at higher concentrations ( > 1 #M) capsaicin exerts non-specific effects on non-sensory nerves and Correspondence to: M. Takaki, Department of Physiology, Okayama University Medical School, Shikatacho, Okayama 700, Japan.
muscle cells (Such and Jancs6, 1986; Barth6 et a!., 1987; Maggi et al., 1987; 1989b). These rL,~dings suggest the presence of a specific pharmacological receptor site in certain afferent nerve endings. The capsaicin-sensitive receptor seems to play an important role in the sensory function of these nerve endings, since capsaicin induces long-lasting insensitivity of these receptors in various tissues (desensitization) (Jancs6 et al., 1967; Szolcsfinyi and Jancs6-G~bor, 1976; Holzer, 1988; Maggi and Meli, 1988). Piperine (piperinoyl-piperidine), the pungent substance in black pepper, is a congener of capsaicin and is about 70 times less active in producing pain than capsaicin. It has been shown
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
72 that pipeline, like capsaicin, can release endogenous substance P in the rat spinal cord (Jhamandas et aL, 1984; Micevych et al., 1983). Recently, it has been reported that piperine, like capsaicin shows chronotropic effects in the isolated spontaneously beating right atria and inotropic effects in the electrically driven left atria of rats, and that these effects are caused by calcitonin gene-related peptide (CGRP) released from non-adrenergic non-cholinergic (NANC) nerves. The development of cross-tachyphylaxis between piperine and capsaicin is due to the depletion of endogenous CGRP (Miyauchi et al., 1988; 1989). Ruthenium red, an inorganic dye which blocks transmembrane Ca 2+ fluxes in neural tissues, ~lectively antagot~es the actions of capsaicin on pe~pheral nerve terminals in the isolated guineapig ileum (Takaki et al., 1989b) as well as in the rat and guinea-pig urinary bladder, rat vas deferens and guinea-pig bronchus and left atria (Maggi et al., 1988a,c; 1989a,c). The aim of the present study was to investigate the effects of piperine on sensory, nerve terminals by exploring whether piperine, like capsaicin, could influence the contractile response to mesenteric nerve stimulation, the twitch response elicited by field stimulation, and spontaneous motility in the isolated guinea-pig ileum. In addition, we investigated whether or not ruthenium red could antagonize the effects of piperine.
2. Materials and methods Adult guinea-pigs of either sex were stunned and exsanguinated. The ileum (except its 15-cm long terminal portion) was used for the experiments. Segments (2.5-3.0 cm) of ile,,..m vAth (or without) the attached mesenteric vessels and nerves were excised and placed in an organ bath containing 15 ml of Krebs solution ~,t 37 o C, and aerated with 5% CO 2 in 02. The composition of the modified Krebs solution was (mM): NaCI 120.9; KC1 5.9; NaHCO 3 14.4; NaH2PO 4 1.2; CaCI 2 2.5; MgC12 1.2 and ~ucose 11.5. Preparations were left to equilibrate for 1.5-2 h. The tissues were treated with guanethJdine (2 ~M) and hexamethonium (50 /tM) before and during the experiments. The
mesenteric vessels and nerves were placed between a pair of annular platinum electrodes for mesenteric nerve stimulation. The stimulation parameters were 20 Hz, 0.5 ms, and supramaximal currents; stimulations were given for 30 s at 5.5rain intervals. Field stimulation was applied by means of two ring platinum electrodes placed around the segment. The stimulation parameters were 0.1 Hz, 0.5 ms and supramaximal currents. The mechanical activity of the longitudinal muscle was recorded with an isotonic transducer; 0.5 g tension was applied. Because the effect of piperine or capsaicin exhibited tachyphylaxis, only one does of piperine or capsaicin per tissue was appliediO~" 15 min. For the study of tachyphylaxis, a second incubation with piperine or capsaicin for 15 min was applied 1 h after the first application. Extrinsic (mesenteric) denervation to induce degeneration of primary afferents was performed 8-10 clays before the experiments, as described by Barth6 and Szolcsfinyi (1978). Drugs used were atropine sulfate (Merck), human calcitonin gene-related peptide (hCGRP; Peptide Institute), capsaicin (Sigma), guanethidine sulfate (Tokyo-Kasei), hexamethonium bromide (Sigma), piperine (Sigma), ruthenium red (Sigma), tetrodotoxin (Sankyo), substance P (Peptide Institute). Stock solutions of piperine (1 and 10 mM) and capsaicin (10 mM) were made up in 100~ dimethyl su!foxide (DMSO); capsaicin was then diluted 1 : 1 0 (1 mM) with distilled water. The stock solutions were diluted 1:1000 with Krebs solution in the organ bath. Thus the final concentration of DMSO was 0.01-0.1~. DMSO had no effect at concentrations less than 1~. All values are presented as means + S.E. Statistical significance of differences between means was estimated with Student's t-test.
3. Results 3.1. Piperine-induced inhibition on the contractile response to mesenteric nerve stimulation
Supramaximal mesenteric nerve stimulation (20 Hz, 0.5 ms for 30 s) evoked a biphasic response (contraction followed by relaxation) in ileal seg-
73 S mm
1st ptperlne 1 IM
[
2nd plperlne 1 uH
S mtn
H
Fig. 1. A typical trace showing the inhibitory effect of piperine (1/LM) on the contractile response of isolated guinea-pig ileum to mesenteric nerve (MN) stimulation (at the dots) in the
presenceof guanethidine(2 ~M) and hexamethonium(50 plVO. (A) First application of piperine (at arrow) for 15 rain. (B) Second applicationof piperine(at arrow) for 15 rain, after an 1-h washoutperiod. W, washout.
xnents after complete blockade of adrenergic neurons with guanethidine (2 #M), which blocks the adrenergic inhibitory respoase to mesenteric nerve stimulation. Hexamethonium (50 #M) was also present in the organ bath to block nicotinic transmission. This drug abolished the effect of nicotine (5 vM), as reported previously by Takaki et al. (1987; 1989a,b). The contractile response to mesenteric nerve stimulation was capsaicin-sensitire, i.e. it was irreversibly and strongly inhibited by capsaicin (1 #M). In the present study, piperine (1 /~M) strongly inhibited the response to res )onse 100 "
50-
O-
LI/ ptpertne 1 ~H
RR 4-
piper'the
I .H
Fig. 2. Ruthenium red (RR, 1 #M) antagonism of the piperine.induced inhibition of the contractile response to mesentefic nerve (MN) stimulation. Open columns, responses obtained in the pre.~ence of piperine. Solid columns, responses after 1 h of washout. * P < 0.02 vs. control (n = 10); * * P < 0.01 vs. control (n =010). Ordinate, • of the contractile response to MN stimulation.
mesenteric nerve stimulation (7.9 + 4.,% of the response remained, n = 10, figs. 1A and 2). One hour after washout of the preparations treated with piper~me for 15 rain, the response to mesenteric nerve stimulation recovered partly (39.3 _+7.3~, n = 9; figs. 1B and 2). Re-administration of piperine (1 ~M) abolished this recovered response (fig. 1B). The inhibitory effect of piperine applied for more than 30 min on the response to mesenteric nerve stimulation persisted for 2 h even after repeated washout. The threshold concentration of piperine for this effect was 0.5 #M. The effects of 1 and 3 #M piperine were not significantly different; therefore, 1 /~M piperine was used to e!;.cit a m~3.mal effect in further experiments. Capsaicin (1 /tM for 15 rain) also strongly inhibited the response to mesenteric nerve stimulation (6.5 _+ 6.5~o response remained, n = 4). However, after 1-h washout, the response had recovered by only 15.2 + 8.8~. The second application of capsaicin abolished this ~esponse.
3.2. Piperine-induced controction and inhibition of the contractile response (twitch) induced by field stimulation Piperine (0.5-10 vM) induced dose-dependent ileal contractions. The amplitude of the contraction evoked by 1 vM piperine was 83.5 + 8.6~ (n = 13) of the maximal twitch response elicited by field stimulation. This contraction was significantly reduced to 36.6 + 11.0~ (n --- 4) by atropine (5 /~M), to 31.9 + 4.5~, (n = 4) by tetrodotoxin (TTX, 0.31 vM) or to 31.6 + 3.15[ (n = 5) by desensitization with substance P. The latter was evoked by a single administration of 0.2/~M s~5stance P according to the method of Takaki et al. (1987). The contractile effect of pipeline (1 VM) was abolished by a combination cf TTX and desensitization with substance P (n = 8). Furthermore, this contraction was abolished by extrinsic denervation (n = 4). 'il~e tachyphylaxis to piperme occurred rapidly depending on the dose (0.5-3.0 #M) used earlier. The contractile effect of piperine (1 vM) was inhibited by repeated application of the d~'ug, although not completely (figs. 1B and 3A). On the other hand, contractions induced by a
high concentration of piperine (10 laM) were not sio~ificantly reduced by repeated application of the drug (fig. 4), iadicating that desensitization did not occur. The contraction induced by 10/~M piperine (149.1 4-_10.9%, n = 12) was partly but significantly reduced to 107.0 + 4.2% (n = 8) by the combination of TTX and desensitization with substance P, or to 77.1 _+ 3.5% (n = 5) by extrinsic denerxation (fig. 4). The inhibitory effect of piperine on the twitch response was studied. After the initial contraction, piperine (1 jaM) produced a prompt and transient inhibition of the twitch contraction (58.0 4-5.3% response remained, n = 13, fig. 3E). Piperine sometimes also caused relaxation after the contraction. In these cases, the twitch contractions were similarly inhibited even after the recovery of the decrease in the basal tone. A second application of piperine 75 rain after the first one hardly had any effect, indicating desensitization (fig. 3E). The inhibitory effect of capsaicin on tl~e twitch
g response 200 !l
100
A
B
C
D
I st p4per| ne 10 uH
A
B
C
D
2nd p~pertne 10 pH
Fig. 4. Effects of tetrodotoxin (TrX, 0.31 FM), desensitization with substance P (SP-D, 0.2 FM), atropine (5 pM) and extrinsic denervation on the contraction induced by piperine (10 pM for 15 min). (A) Control (n =12). (B) Effects of TTX plus SP-D (n--8). (C) Effects of atropine, TTX and SP-D (n = 4). (D) Effects of extrinsic denervation (n = 5). Piperine (10/tM) was applied for the second time 1 h after washout. * Significandy (P < 0.005) different from the control (A). The lack of a significant difference between the responses to the first and second application of drug indicated that there was no desensitization.
response
0
1st 2ml
1st 2ad p|p ptp
1st 2nd ptp cap
Ist 2nd ptp cap
1st 2nd _cap pip
Ist 2nd cap ptp
Ist 2nd cap c~ap
i "!i!* 1st 2nd cap cap
Fig. 3. Cross-tachyphylaxis between piperine (pip) and capsaicin (cap). (A-D) The contractile response to piperine (1/~M) or capsaicin (1 IaM) expressed as ~ of the maximal twitch contractions induced by field stimulation. (E-H) The inhibition of the twitch contractions evoked by pipeline (1/~M) or capsaicin (1 ~tM) expressed as % response of the pre-drug twitch. Open column, the response induced by the application of pipefine or capsaicin as the first drug; dotted column, the response induced by the application of piperine or capsaicm as the second drug. * Significantly different from the response to the first drag appfication. Number of experiments, n = 5-8 in (A) through (H~.
response is probably mediated via the release of calcitonin gene-related peptide (CGRP) from sensory nerve endings (Barth6 et al., 1987; Maggi et al., 1988b). In the present study, the inhibitory effec*, of pipefine on the twitch response was significantly antagonized by desensitization w;th CGRP (90.1 + 2.7% of the twitch response persisted, n - - 6 ) . Desensitization was attained by applying CGRP (25 nM) for 2 × 10 min according to the method described by Takaki et al. (1989a); it did not effect the piperine-induced contraction. Piperine (10 /~M) abolished the twitch response and reduced the contraction induced by substance P (0.1/tM) to 51.2 + 8.4% of the control (n = 5). In the extrinsically denervated loops, this inhibitory effect was not changed and failed to show tachyphylaxis (data not shown).
3.3 Antagonism of the effects of piperine by ruthenium red The tissues were exposed to ruthenium red (1 /~M) for 25 min before exposure to piperine (1 /LM), as described previously by Takaki et al. (1989b). Ruthenium red itself sometimes increased
75
5 mm
A
------RR 1 ~H ~
mtn ~']st ptpertne | UH
t W
2rid ptpertne 1 .N Fig. 5. Typical trace showing ruthenium red (RR) antagonism of the piperine-induced inhibition of the contractile response to mesenteric nerve (MN) stimulation (at the dots) in the presence of guanethidine (2 ltM) and hexamethonium (50/tM). (A) Responses in the presence of RR (1 ttM) administered 25 min before piperine; (B) responses after 1-h washout.
the basal tone of the ileum, but in many cases it did not affect the basal tone or the response to mesenteric nerve stimulation per se, as shown in fig. 5A. It has been reported that ruthenium red (1 /~M) selectively antagonizes the contraction and inhibition of the response to mesenteric nerve stimulation evoked by capsaicin (1/tM) (Takaki et al., 1989b). Ruthenium red (1 /~M) antagonized the inhibitory effect of piperine (1 /~M) on the response elicited by mesenteric nerve stimulation (fig. 5A); the response remaining after piperine was 37.7 + 6.5% ( n - 1 0 ) (fig. 2). After l-h washout, the response to mesenteric nerve stimulation had recovered to a significantly (P < 0.005) greater extent than that in the control loops (figs. 1B, 2 and 5B). Ruthenium red (1 ~M) also significantly reduced (P < 0.005) the contraction induced by piperine (1 /~M) to 36.2 4-8.7% ( n - 1 0 ) . Ruthenium red (1-10/~M) had a marked inhibitory effect (40-100%) on the twitch response per se, and for this reason a concentration of 0.5 ttM was selected for further experiments. This concentration of ruthenium red did not affect the twitch response (92.4 4- 4.1%, n ffi 7) and antagonized the inhibitory effect of capsaicin (1 /~M) on the twitch response without exerting effects on the CGRP-induced inhibition of the ~witch, as reported previously (Takaki et al., 1989b). In the present study, ruthenium red (0.5
/LM) antagonized the inhibition of the twitch response evoked by piperine (1 tiM), without having an effect on the twitch per se (data not sbown). Piperine (1 tiM) reduced the twitch response from 100 to 61.5 4. 5.7% ( n - 8) in untreated preparations, but only to 80.6 + 4.6% in the presence of ruthenium red (n = 10).
3.4. Cross-tachyphylaxis between piperine and capsaicin After the first application of piperine (1 /~M) for 15 min and washout for 1 h, the second application of piperine caused a small contraction (fig. 3A) and hardly had any effect on the twitch (fig. 3E); the application of capsaicin (1 ttM) was haeffective (fig. 3B and F). After the first application of capsaicin (1/zM) for 15 rain and washout for 1 h, the appb_'cation of piperine (1/zM) caused a small contraction and no effects on the twitch (fig. 3C and G); the second application of capsaicin (1/tM) did not have an effect (fig. 3D and H).
4. Discussion
The present findings on the motility of the guinea-pig ileum indicated that the effects of piperine were similar to those of capsaicin (Barth6 and Szolcshnyi, 1978; Szolcshnyi and Barth6, 1978: Barth6 et al., 1982; Chahl, 1982; Barth6 et al, 1987; Maggi zt ~., 1988b; Takaki et al., 1987, 1989a,b,c; Takaki and Nakayama, 1989). The desensitization induced by piperine (1/tM) may be due to a mechanism similar to that of capsaicin, i.e. depletion of a releasable pool of sensory neuropeptides, such as substance P a n d / o r CGRP, excessive entry of Ca 2+ into sensory neurons, as well as other mechanisms (Szolcshnyi, 1985; Holzer, 1988; Gamse et al., 1979; Maggi et al., 1988c; Dray et al., 1989; Jancs6 et al., 1984; Santicioli et al., 1987; Marsh et al., 1987; Wood et al., 1988). It has been shown that piperine reduces the level of substance P in the rat spinal cord, probably as a result of an extensive release of" this neuropeptide (Jhamandas et al., 1984; Micevych et al., 1983). From the present results, the con-
76
tractile effect of piperine (1 /tM) is thought to involve the release of substance P from sensory ,¢~'es" the released substance P acts ph-tly via ~ , . : a , 0 cholinergic neurons and partly on the muscle d/rectly. About 50% of contraction induced by the high concentration (10 /tM) of piperine was due to a non-specific direct action on the smooth muscle i ~ . linis non-specifi~ effect of piperine is quite distinct from that of capsaicin (Barth6 et al., 1987; Maggi et al., 1987; 1989b). The inhibitory effect of capsaicin on the twitch response is thought to involve the release of endogenous CGRP from sensory nerves (Barth6 et al., 1987; Maggi et al., 1988c). It has been shown that piperine, like capsaicin, releases and depletes CGRP from intracardiac NANC ner,~es, and cross-tachyphylaxis between piperine and capsaicin exists (Miyauchi et al., 1988; 1989). The inhibitory effect of low concentrations of piperine (1 iaM) on the twitch observed in this study probably involves the release of endogenous CGRP from sensory nerves. However, the inhibitory effect of piperine (10 laM) is thought to be non-specific, similar tO that of high concentrations of capsaicin (Such and Jancs6, 1986; Barth6 et al., 1987; Maggi et al., 1987; 1989b). Recently, it has been reported that ruthenium red selectively antagonizes capsaicin-induced actions (Maggi et al., 1988a; 1989a; Takaki et al., 1989b) and inhibits capsaicin-induced transmitter release in the urinary bladder, sm~J! intestine, vas deferens, bronchus and left atria (Maggi et al., 1988c; 1989c). However, ruthenium red also inhibits afferent responses to capsaicin that do not involve local transndtter release (Amann and LembecL 1989~. In the present study, ruthenium red antagonized the three effects of piperine and protected against desensitization. The mechanism for this antagonism is not known at present, although ruthenium red could act at a capsaicinactivated cation channel as proposed by Chahl (1989) and Amann et al. (1989). On the other hand, the application of piperine (1 /tM) as the second drug caused a small contractile response, irrespective of whetber capsaicin or piperine was the drug applied first. The contractile response induced by piperine (1/zM) was probably the result of specific effect on sensory
nerves. It seems likely that the effects of pipeline are less susceptible to desensitization than those of capsaicin. We conclude that piperine caused its effects by a mechanism, similar to that of capsaicin, which possibly involves bin :d:.ng m a common specific pharmacological receptor site in afferent nerve endings. Ruthenium red seems to be a promising specific antagonist of these pungent agents.
References Amann, R., J. Donnerer and F. Lembeck, 1989, Capsaicin-induced stimulation of polymodal nociceptors is antagonized by ruthenium red independently of extracellular calcium, Neuroscience 32, 255. Amann, R. and F. Lembeck, 1989, Ruthenium red selectively prevents capsaicin-induced nociceptor stimulation, European J. Pharmacol. 161, 227. Barth6, L., P. Holzer, F. Lembeck and J. Szolcs~nyi, 1982, Evidence that the contractile response of the gninea-pig ileum to capsaicin is due to release of substance P, J. Physiol. 332, 157. Barth6, L., G. Peth6, A. Antal, P. Holzer and J. Szolcs~ayi, 1987, Two types of relaxation due to capsaicin in the guinea pig isolated ileum, Neurosci. Lett. 81, 146. Barth6, L. and J. Szolcs~nyi, 1978, The site of action of capsaicin on the guinea-pig isolated ileum, NaunynSchmiedeb. Arch. Pharmacol. 305, 75. Chahl, L.A., 1982, Evidence that the contractile response of the guinea-pig ileum to capsaicin is due to substance P release, Naunyn-Schmiedeb. Arch. Pharmacol. 319, 212. Chahl, L.A., 1989, The effects of ruthenium red on the response of guinea-pig ileum to capsaicin, European J. Pharmacol. 169, 241. Dray, A., M.W. Hankins and J.C. Yeats, 1989, Desensitization and capsaicin-induced release of substance P-like immunoreactivity from guinea-pig ureter in vi~o, Neuroscience 31, 479. Gamse, R., A. Moln~r and F. Lembeck, 1979, Substance P release from spinal cord slices by capsaicin, Life Sci. 25, 629. Holzer, P., 1988, Local effector function of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides, Neuroscience 24, 739. Jancs6, G., S. Karcsu, E. Kiraly, A. Szebeni, L. Toth, E. Bacsy, F. Joo and A. Parducz, 1984, Neurotoxin-induced nerve cell degeneration: possible involvement of calcium, Brain Res. 295, 211. Jancsb, N., A. Jancsb-G~ibor and J. Szolcsb,nyi, 1967, Direct evidence for neurogenic inflammation and its prevention by denervation and by pretreatment with capsaicin, Br. J. Pharmacol. 31, 138.
77 Jhamandas, K., T.L. Yaksh, G. Harty, J. Szolcs~nyi and V.L. Go, 1984, 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, 215. Maggi, C.A., S. Giuliani and A. Meli, 1989a, Effect of ruthenium red on responses mediated by activation of capsaicin-sensitive nerves of the rat urinary bladder, Naunyn-Schmiedeb. Arch. Pharmacol. 340, 541. Maggi, C.A., l.Th. Lippe, S. Giuliani, L. Abeili, V. Somma, P. Geppetti, G. Jancs6, P. Santicioli and A. Meli, 1989b, Topical versus systemic capsaicin desensitization: specific and unspecific effects as indicated by w,~lificafion or reflex micturition in rats, Neuroscience 31, 745. Maggi, C.A. a~.,d A. Meli, 1988, The sensory-efferent function of capsaicin-sensitive nerves, Gen. Pharmacol. 19, 1. Maggi, C.A., A. Mefi and P. Santicioli, 1987, Four motor effects of capsaicin on guinea-pig distal colon, Br. J. Pharmacol. 90, 651. Maggi, C.A., R. Patacchini, P. Santicioli, S. Giuliani, E.D. Bianco, P. Geppetti and A. Meli, 1989c, The "efferent' function of capsaicm-sensitive nerves: Ruthenium Red discriminates between different mechanisms of activation, European J. Pharmacol. 170, 167. Maggi, C.A., R. Patacchini, P. Santicioli, S. Giuliani, P. Geppetti and A. Meli, 1988a, Protective action of ruthenium red toward capsaicin desensitization of sensory fibers, Neurosci. Lett. 88, 201. Maggi, C.A., R. Patacchini, P. Santicioli, E. Theodorsson and A. Meli, 1988b, Several neuropeptides determine the vi~J~eromotor response to capsaicin in the guinea-pig isola~.ed ileal longitudinal muscle, European J. Pharmacol. 148, 43. Maggi, C.A., P. Santicioli, P. Geppetti, M. Parlani, M. Astolfi, P. Pradelles, R. Patacchini and A. Meli, 1988c, The antagonism induced by Ruthenium Red of the actions of capsaicin on the peripheral terminals of sensory neurons: further studies, European J. Pharmacoi. 154, 1. Marsh, S.$., C.E. Stansfeld, D.A. Brown, R. Davey and D. McCarthy, 1987, The mechanism of action of capsaicin on sensory C-type neurons and their axons in vitro, Neuroscience 23, 275. Mice,~,ch, P.E., T.L. Yaksh and J. Szolcsb.nyi, 1983, Effects of intrathecal capsaicin analogues on the immunofluorescence of peptides and serotonin in the dorsal horn in rats, Neuro~..oien~ 8, 123. Miyauchi, T., T. Ishikawa, Y. Sugishita, A. Saho and K. Goto, 1988, Effects of piperine on calcitonin gene-related peptide (CGRP)-containing nerves !tn the isolated rat atria, Neurosci. Lett. 91, 222. Miyauchi, T., T. Ishikawa, Y. Sugishita, A. Saito and K. Goto,
1989, Involvement of calcitonin gene-related peptide in the positive chronotropic and inotropic effects of piperine and development of cross-tachyphylaxis between piperine and capsaicin in the isolated rat atria, J. Pharmacol. Exp. Ther. 248, 816. Santicioli, P., R. Pataochini, C.A. Maggi and A. Meli, 1987, Exposure to ealcium-free medium protects sensory fibers by capsaicin desensitization, Neurosci. Lett. 80, 167. Such, G. and G. Jancs6, 1986, Axonal effects of capsaicin: an electrophysiological study, Acta Physiol. Hung. 67, 53. Szolcsinyi, J., 1985, Sensory receptors and the antinociceptive effects of capsaicin, in: Tachykinin Antagonists, eds. IL H~kanson and F. $ ~ d l e r (Elsevier S~iel~c,ePubfishers B.V., Amsterdam) p. 45. Szolcdmyi, J. and L. Barth6, 1978, New type of nerve-mediated cholinergic contractions of the guinea-pig small intestine and its selective blockade by capsaicin, NaunynSchmiedeb. Arch. Pharmacol. 305, 83. Szolcs~nyi, J. and A. Jancsb-G~ibor, 1975, Sensory effects of capsaicin congeners. I. Relationship between chemical structure and pain-producing potency of pungent agents, Arzneim. Forsch. (Drug Res.) 25, 1877. Szolcs~myi, J. and A. Jancsb-G,~ibor, 1976, Sensory effects of capsaicin congeners. Part II: Importance of chemical structure and pungency in desensitizing activity of capsaicin-type compounds, Arzneim. Forsch. (Drug Res.) 26, 33. Takaki, M., J.-G. Jin and S. Nakayama, 1987, Involvement of hexamethonium-sensitive and capsaicin-sensitive components in cholinergic contractions induced by mesenteric nerve stimulation in guinea-pig ileum, Biomed. Res. 8, 195. Takaki, M., J.-G. Jin and S. Nakayama, 1989a, Possible involvement of calcitonin gene-related peptide (CGRP) in non-cholinergic non-adrenergic relaxation induced by mesenteric nerve stimulation in guinea pig ileum, Brain Res. 478, 199. Takaki, M.. I.-G. Jin and S. Nakayama, 1989b, Ruthenium red antagonism of the effect of capsaicin on the motility of the isolated guinea-pig ileum, European J. Pharmacol. 174, 57. Takaki, M., J.G. Jin and S. Nal~=ayama, 1989c, Effects of capsaicin on the circular muscle motility of the isolated guinea-pig ileum, Acta Med. Okayama, 43, 353. Takaki, M. and S. Nakayama, 1989, Effects of capsaicin on myenteric neurons of the guinea pig ileum, Neurosci. Lett. 105, 125. Toh, C.C., T.S. Lee and A.K. Kiang, 1955, The pharmacological actions of capsaicin and analogues, Br. J. Pharmacol. 10, !75. Wood, J.lq., J. Winter, I.F. James, H.P. Rang, J. Yeats and S. Bevan, 1988, Capsaicin-induced ion fluxes in dorsal root ganglion cells in culture, J. Neurosci. 8, 3208.
71
EJP 51469
Effects of pipeline on the motility of the isolated guinea-pig ileum: comparison with capsaicin M i y a k o Takaki, J i - G u a n g Jill, Y u n - F e i L u a n d Sosogu N a k a y a r n a Department of Physiology, Okayama University Medical School, Shikatacho, Okayama 700, Japan Received 30 October 1989,revised MS received 29 May 1990, accepted 19 June 1990
Piperine (1 gM), a congener of capsaicin, produced an initial contraction, blocked the capsaicin-sensitive contractile response to mesenteric nerve stimulation and inhibited the twitch response induced by field stimulation in the isolated guinea-pig ileum. These three effects of piperine (1 /zM) were rapidly desensitized and significantly antagonized by ruthenium red (0.5-1 /zM), an inorganic dye known to antagonize the effects of capsaicin. The contractile effect of piperine was abolished by application of tetrodotoxin plus desensitization with substance P or by extrinsic denervation. The inhibitory effect of piperine (1/tM) on the twitch response was antagonized by desensitization with calcitonin gene-related peptide (CGRP). Moreover, cross-tachyphylaxis between piperine and capsaicin was observed, suggesting that a similar mechanism may be involved in the effects of these agents. The contractile effects induced by piperine (10/tM) and the subsequent inhibitory effects on the twitch response were not desensitized and largely persisted after extrinsic denervation. The contractile effects of piperine (10 pM) were not strongly inhibited by tetrodotoxin plus desensitization with substance P. It was concluded that the lower concentration of piperine caused contraction and inhibited the twitch responses by releasing substance P arid CGRP, respectively, from sensory nerves, and blocked the response to mesenteric nerve stimulation by a mechanism similar to that of capsaicin. At higher concentrations, piperine had non-specific direct actions on the smooth muscle. Piperine; Capsaicin; Calcitonin gene-related peptide (CGRP); Substance P; Ruthenium red; Ileal motility; (Guinea-pig)
1. Introduction
The stimulating effect of capsaicin, the pungent substance in red pepper, is specific for chemosensitive sensory nerves at low concentrations (1/~M) and there is no evidence that it has a direct effect on efferent neuronal structures (Toh et al., 1955; Jancs6 et al., 1967; Szolcsfinyi and Jancs6-G~bor, 1975; Barth6 and Szolcsfinyi, 1978), although at higher concentrations ( > 1 #M) capsaicin exerts non-specific effects on non-sensory nerves and Correspondence to: M. Takaki, Department of Physiology, Okayama University Medical School, Shikatacho, Okayama 700, Japan.
muscle cells (Such and Jancs6, 1986; Barth6 et a!., 1987; Maggi et al., 1987; 1989b). These rL,~dings suggest the presence of a specific pharmacological receptor site in certain afferent nerve endings. The capsaicin-sensitive receptor seems to play an important role in the sensory function of these nerve endings, since capsaicin induces long-lasting insensitivity of these receptors in various tissues (desensitization) (Jancs6 et al., 1967; Szolcsfinyi and Jancs6-G~bor, 1976; Holzer, 1988; Maggi and Meli, 1988). Piperine (piperinoyl-piperidine), the pungent substance in black pepper, is a congener of capsaicin and is about 70 times less active in producing pain than capsaicin. It has been shown
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
72 that pipeline, like capsaicin, can release endogenous substance P in the rat spinal cord (Jhamandas et aL, 1984; Micevych et al., 1983). Recently, it has been reported that piperine, like capsaicin shows chronotropic effects in the isolated spontaneously beating right atria and inotropic effects in the electrically driven left atria of rats, and that these effects are caused by calcitonin gene-related peptide (CGRP) released from non-adrenergic non-cholinergic (NANC) nerves. The development of cross-tachyphylaxis between piperine and capsaicin is due to the depletion of endogenous CGRP (Miyauchi et al., 1988; 1989). Ruthenium red, an inorganic dye which blocks transmembrane Ca 2+ fluxes in neural tissues, ~lectively antagot~es the actions of capsaicin on pe~pheral nerve terminals in the isolated guineapig ileum (Takaki et al., 1989b) as well as in the rat and guinea-pig urinary bladder, rat vas deferens and guinea-pig bronchus and left atria (Maggi et al., 1988a,c; 1989a,c). The aim of the present study was to investigate the effects of piperine on sensory, nerve terminals by exploring whether piperine, like capsaicin, could influence the contractile response to mesenteric nerve stimulation, the twitch response elicited by field stimulation, and spontaneous motility in the isolated guinea-pig ileum. In addition, we investigated whether or not ruthenium red could antagonize the effects of piperine.
2. Materials and methods Adult guinea-pigs of either sex were stunned and exsanguinated. The ileum (except its 15-cm long terminal portion) was used for the experiments. Segments (2.5-3.0 cm) of ile,,..m vAth (or without) the attached mesenteric vessels and nerves were excised and placed in an organ bath containing 15 ml of Krebs solution ~,t 37 o C, and aerated with 5% CO 2 in 02. The composition of the modified Krebs solution was (mM): NaCI 120.9; KC1 5.9; NaHCO 3 14.4; NaH2PO 4 1.2; CaCI 2 2.5; MgC12 1.2 and ~ucose 11.5. Preparations were left to equilibrate for 1.5-2 h. The tissues were treated with guanethJdine (2 ~M) and hexamethonium (50 /tM) before and during the experiments. The
mesenteric vessels and nerves were placed between a pair of annular platinum electrodes for mesenteric nerve stimulation. The stimulation parameters were 20 Hz, 0.5 ms, and supramaximal currents; stimulations were given for 30 s at 5.5rain intervals. Field stimulation was applied by means of two ring platinum electrodes placed around the segment. The stimulation parameters were 0.1 Hz, 0.5 ms and supramaximal currents. The mechanical activity of the longitudinal muscle was recorded with an isotonic transducer; 0.5 g tension was applied. Because the effect of piperine or capsaicin exhibited tachyphylaxis, only one does of piperine or capsaicin per tissue was appliediO~" 15 min. For the study of tachyphylaxis, a second incubation with piperine or capsaicin for 15 min was applied 1 h after the first application. Extrinsic (mesenteric) denervation to induce degeneration of primary afferents was performed 8-10 clays before the experiments, as described by Barth6 and Szolcsfinyi (1978). Drugs used were atropine sulfate (Merck), human calcitonin gene-related peptide (hCGRP; Peptide Institute), capsaicin (Sigma), guanethidine sulfate (Tokyo-Kasei), hexamethonium bromide (Sigma), piperine (Sigma), ruthenium red (Sigma), tetrodotoxin (Sankyo), substance P (Peptide Institute). Stock solutions of piperine (1 and 10 mM) and capsaicin (10 mM) were made up in 100~ dimethyl su!foxide (DMSO); capsaicin was then diluted 1 : 1 0 (1 mM) with distilled water. The stock solutions were diluted 1:1000 with Krebs solution in the organ bath. Thus the final concentration of DMSO was 0.01-0.1~. DMSO had no effect at concentrations less than 1~. All values are presented as means + S.E. Statistical significance of differences between means was estimated with Student's t-test.
3. Results 3.1. Piperine-induced inhibition on the contractile response to mesenteric nerve stimulation
Supramaximal mesenteric nerve stimulation (20 Hz, 0.5 ms for 30 s) evoked a biphasic response (contraction followed by relaxation) in ileal seg-
73 S mm
1st ptperlne 1 IM
[
2nd plperlne 1 uH
S mtn
H
Fig. 1. A typical trace showing the inhibitory effect of piperine (1/LM) on the contractile response of isolated guinea-pig ileum to mesenteric nerve (MN) stimulation (at the dots) in the
presenceof guanethidine(2 ~M) and hexamethonium(50 plVO. (A) First application of piperine (at arrow) for 15 rain. (B) Second applicationof piperine(at arrow) for 15 rain, after an 1-h washoutperiod. W, washout.
xnents after complete blockade of adrenergic neurons with guanethidine (2 #M), which blocks the adrenergic inhibitory respoase to mesenteric nerve stimulation. Hexamethonium (50 #M) was also present in the organ bath to block nicotinic transmission. This drug abolished the effect of nicotine (5 vM), as reported previously by Takaki et al. (1987; 1989a,b). The contractile response to mesenteric nerve stimulation was capsaicin-sensitire, i.e. it was irreversibly and strongly inhibited by capsaicin (1 #M). In the present study, piperine (1 /~M) strongly inhibited the response to res )onse 100 "
50-
O-
LI/ ptpertne 1 ~H
RR 4-
piper'the
I .H
Fig. 2. Ruthenium red (RR, 1 #M) antagonism of the piperine.induced inhibition of the contractile response to mesentefic nerve (MN) stimulation. Open columns, responses obtained in the pre.~ence of piperine. Solid columns, responses after 1 h of washout. * P < 0.02 vs. control (n = 10); * * P < 0.01 vs. control (n =010). Ordinate, • of the contractile response to MN stimulation.
mesenteric nerve stimulation (7.9 + 4.,% of the response remained, n = 10, figs. 1A and 2). One hour after washout of the preparations treated with piper~me for 15 rain, the response to mesenteric nerve stimulation recovered partly (39.3 _+7.3~, n = 9; figs. 1B and 2). Re-administration of piperine (1 ~M) abolished this recovered response (fig. 1B). The inhibitory effect of piperine applied for more than 30 min on the response to mesenteric nerve stimulation persisted for 2 h even after repeated washout. The threshold concentration of piperine for this effect was 0.5 #M. The effects of 1 and 3 #M piperine were not significantly different; therefore, 1 /~M piperine was used to e!;.cit a m~3.mal effect in further experiments. Capsaicin (1 /tM for 15 rain) also strongly inhibited the response to mesenteric nerve stimulation (6.5 _+ 6.5~o response remained, n = 4). However, after 1-h washout, the response had recovered by only 15.2 + 8.8~. The second application of capsaicin abolished this ~esponse.
3.2. Piperine-induced controction and inhibition of the contractile response (twitch) induced by field stimulation Piperine (0.5-10 vM) induced dose-dependent ileal contractions. The amplitude of the contraction evoked by 1 vM piperine was 83.5 + 8.6~ (n = 13) of the maximal twitch response elicited by field stimulation. This contraction was significantly reduced to 36.6 + 11.0~ (n --- 4) by atropine (5 /~M), to 31.9 + 4.5~, (n = 4) by tetrodotoxin (TTX, 0.31 vM) or to 31.6 + 3.15[ (n = 5) by desensitization with substance P. The latter was evoked by a single administration of 0.2/~M s~5stance P according to the method of Takaki et al. (1987). The contractile effect of pipeline (1 VM) was abolished by a combination cf TTX and desensitization with substance P (n = 8). Furthermore, this contraction was abolished by extrinsic denervation (n = 4). 'il~e tachyphylaxis to piperme occurred rapidly depending on the dose (0.5-3.0 #M) used earlier. The contractile effect of piperine (1 vM) was inhibited by repeated application of the d~'ug, although not completely (figs. 1B and 3A). On the other hand, contractions induced by a
high concentration of piperine (10 laM) were not sio~ificantly reduced by repeated application of the drug (fig. 4), iadicating that desensitization did not occur. The contraction induced by 10/~M piperine (149.1 4-_10.9%, n = 12) was partly but significantly reduced to 107.0 + 4.2% (n = 8) by the combination of TTX and desensitization with substance P, or to 77.1 _+ 3.5% (n = 5) by extrinsic denerxation (fig. 4). The inhibitory effect of piperine on the twitch response was studied. After the initial contraction, piperine (1 jaM) produced a prompt and transient inhibition of the twitch contraction (58.0 4-5.3% response remained, n = 13, fig. 3E). Piperine sometimes also caused relaxation after the contraction. In these cases, the twitch contractions were similarly inhibited even after the recovery of the decrease in the basal tone. A second application of piperine 75 rain after the first one hardly had any effect, indicating desensitization (fig. 3E). The inhibitory effect of capsaicin on tl~e twitch
g response 200 !l
100
A
B
C
D
I st p4per| ne 10 uH
A
B
C
D
2nd p~pertne 10 pH
Fig. 4. Effects of tetrodotoxin (TrX, 0.31 FM), desensitization with substance P (SP-D, 0.2 FM), atropine (5 pM) and extrinsic denervation on the contraction induced by piperine (10 pM for 15 min). (A) Control (n =12). (B) Effects of TTX plus SP-D (n--8). (C) Effects of atropine, TTX and SP-D (n = 4). (D) Effects of extrinsic denervation (n = 5). Piperine (10/tM) was applied for the second time 1 h after washout. * Significandy (P < 0.005) different from the control (A). The lack of a significant difference between the responses to the first and second application of drug indicated that there was no desensitization.
response
0
1st 2ml
1st 2ad p|p ptp
1st 2nd ptp cap
Ist 2nd ptp cap
1st 2nd _cap pip
Ist 2nd cap ptp
Ist 2nd cap c~ap
i "!i!* 1st 2nd cap cap
Fig. 3. Cross-tachyphylaxis between piperine (pip) and capsaicin (cap). (A-D) The contractile response to piperine (1/~M) or capsaicin (1 IaM) expressed as ~ of the maximal twitch contractions induced by field stimulation. (E-H) The inhibition of the twitch contractions evoked by pipeline (1/~M) or capsaicin (1 ~tM) expressed as % response of the pre-drug twitch. Open column, the response induced by the application of pipefine or capsaicin as the first drug; dotted column, the response induced by the application of piperine or capsaicm as the second drug. * Significantly different from the response to the first drag appfication. Number of experiments, n = 5-8 in (A) through (H~.
response is probably mediated via the release of calcitonin gene-related peptide (CGRP) from sensory nerve endings (Barth6 et al., 1987; Maggi et al., 1988b). In the present study, the inhibitory effec*, of pipefine on the twitch response was significantly antagonized by desensitization w;th CGRP (90.1 + 2.7% of the twitch response persisted, n - - 6 ) . Desensitization was attained by applying CGRP (25 nM) for 2 × 10 min according to the method described by Takaki et al. (1989a); it did not effect the piperine-induced contraction. Piperine (10 /~M) abolished the twitch response and reduced the contraction induced by substance P (0.1/tM) to 51.2 + 8.4% of the control (n = 5). In the extrinsically denervated loops, this inhibitory effect was not changed and failed to show tachyphylaxis (data not shown).
3.3 Antagonism of the effects of piperine by ruthenium red The tissues were exposed to ruthenium red (1 /~M) for 25 min before exposure to piperine (1 /LM), as described previously by Takaki et al. (1989b). Ruthenium red itself sometimes increased
75
5 mm
A
------RR 1 ~H ~
mtn ~']st ptpertne | UH
t W
2rid ptpertne 1 .N Fig. 5. Typical trace showing ruthenium red (RR) antagonism of the piperine-induced inhibition of the contractile response to mesenteric nerve (MN) stimulation (at the dots) in the presence of guanethidine (2 ltM) and hexamethonium (50/tM). (A) Responses in the presence of RR (1 ttM) administered 25 min before piperine; (B) responses after 1-h washout.
the basal tone of the ileum, but in many cases it did not affect the basal tone or the response to mesenteric nerve stimulation per se, as shown in fig. 5A. It has been reported that ruthenium red (1 /~M) selectively antagonizes the contraction and inhibition of the response to mesenteric nerve stimulation evoked by capsaicin (1/tM) (Takaki et al., 1989b). Ruthenium red (1 /~M) antagonized the inhibitory effect of piperine (1 /~M) on the response elicited by mesenteric nerve stimulation (fig. 5A); the response remaining after piperine was 37.7 + 6.5% ( n - 1 0 ) (fig. 2). After l-h washout, the response to mesenteric nerve stimulation had recovered to a significantly (P < 0.005) greater extent than that in the control loops (figs. 1B, 2 and 5B). Ruthenium red (1 ~M) also significantly reduced (P < 0.005) the contraction induced by piperine (1 /~M) to 36.2 4-8.7% ( n - 1 0 ) . Ruthenium red (1-10/~M) had a marked inhibitory effect (40-100%) on the twitch response per se, and for this reason a concentration of 0.5 ttM was selected for further experiments. This concentration of ruthenium red did not affect the twitch response (92.4 4- 4.1%, n ffi 7) and antagonized the inhibitory effect of capsaicin (1 /~M) on the twitch response without exerting effects on the CGRP-induced inhibition of the ~witch, as reported previously (Takaki et al., 1989b). In the present study, ruthenium red (0.5
/LM) antagonized the inhibition of the twitch response evoked by piperine (1 tiM), without having an effect on the twitch per se (data not sbown). Piperine (1 tiM) reduced the twitch response from 100 to 61.5 4. 5.7% ( n - 8) in untreated preparations, but only to 80.6 + 4.6% in the presence of ruthenium red (n = 10).
3.4. Cross-tachyphylaxis between piperine and capsaicin After the first application of piperine (1 /~M) for 15 min and washout for 1 h, the second application of piperine caused a small contraction (fig. 3A) and hardly had any effect on the twitch (fig. 3E); the application of capsaicin (1 ttM) was haeffective (fig. 3B and F). After the first application of capsaicin (1/zM) for 15 rain and washout for 1 h, the appb_'cation of piperine (1/zM) caused a small contraction and no effects on the twitch (fig. 3C and G); the second application of capsaicin (1/tM) did not have an effect (fig. 3D and H).
4. Discussion
The present findings on the motility of the guinea-pig ileum indicated that the effects of piperine were similar to those of capsaicin (Barth6 and Szolcshnyi, 1978; Szolcshnyi and Barth6, 1978: Barth6 et al., 1982; Chahl, 1982; Barth6 et al, 1987; Maggi zt ~., 1988b; Takaki et al., 1987, 1989a,b,c; Takaki and Nakayama, 1989). The desensitization induced by piperine (1/tM) may be due to a mechanism similar to that of capsaicin, i.e. depletion of a releasable pool of sensory neuropeptides, such as substance P a n d / o r CGRP, excessive entry of Ca 2+ into sensory neurons, as well as other mechanisms (Szolcshnyi, 1985; Holzer, 1988; Gamse et al., 1979; Maggi et al., 1988c; Dray et al., 1989; Jancs6 et al., 1984; Santicioli et al., 1987; Marsh et al., 1987; Wood et al., 1988). It has been shown that piperine reduces the level of substance P in the rat spinal cord, probably as a result of an extensive release of" this neuropeptide (Jhamandas et al., 1984; Micevych et al., 1983). From the present results, the con-
76
tractile effect of piperine (1 /tM) is thought to involve the release of substance P from sensory ,¢~'es" the released substance P acts ph-tly via ~ , . : a , 0 cholinergic neurons and partly on the muscle d/rectly. About 50% of contraction induced by the high concentration (10 /tM) of piperine was due to a non-specific direct action on the smooth muscle i ~ . linis non-specifi~ effect of piperine is quite distinct from that of capsaicin (Barth6 et al., 1987; Maggi et al., 1987; 1989b). The inhibitory effect of capsaicin on the twitch response is thought to involve the release of endogenous CGRP from sensory nerves (Barth6 et al., 1987; Maggi et al., 1988c). It has been shown that piperine, like capsaicin, releases and depletes CGRP from intracardiac NANC ner,~es, and cross-tachyphylaxis between piperine and capsaicin exists (Miyauchi et al., 1988; 1989). The inhibitory effect of low concentrations of piperine (1 iaM) on the twitch observed in this study probably involves the release of endogenous CGRP from sensory nerves. However, the inhibitory effect of piperine (10 laM) is thought to be non-specific, similar tO that of high concentrations of capsaicin (Such and Jancs6, 1986; Barth6 et al., 1987; Maggi et al., 1987; 1989b). Recently, it has been reported that ruthenium red selectively antagonizes capsaicin-induced actions (Maggi et al., 1988a; 1989a; Takaki et al., 1989b) and inhibits capsaicin-induced transmitter release in the urinary bladder, sm~J! intestine, vas deferens, bronchus and left atria (Maggi et al., 1988c; 1989c). However, ruthenium red also inhibits afferent responses to capsaicin that do not involve local transndtter release (Amann and LembecL 1989~. In the present study, ruthenium red antagonized the three effects of piperine and protected against desensitization. The mechanism for this antagonism is not known at present, although ruthenium red could act at a capsaicinactivated cation channel as proposed by Chahl (1989) and Amann et al. (1989). On the other hand, the application of piperine (1 /tM) as the second drug caused a small contractile response, irrespective of whetber capsaicin or piperine was the drug applied first. The contractile response induced by piperine (1/zM) was probably the result of specific effect on sensory
nerves. It seems likely that the effects of pipeline are less susceptible to desensitization than those of capsaicin. We conclude that piperine caused its effects by a mechanism, similar to that of capsaicin, which possibly involves bin :d:.ng m a common specific pharmacological receptor site in afferent nerve endings. Ruthenium red seems to be a promising specific antagonist of these pungent agents.
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