High-Concentration Piperine: Capsaicin-Sensitive and -Insensitive Effects on Isolated Organs

Timea Bencsik Zsolt Sandor Lorand Bartho
Department of Pharmacology and Pharmacotherapy, University of Pécs Medical School, Pécs, Hungary

Key Words : Piperine · Guinea-pig · Ileum · Bladder · Trachea · Contraction · TRPV1 · TRPA1

Abstract

Piperine (P), a sensory stimulant in black pepper, is an ago- nist on TRPV1 receptors. Earlier work has showed capsaicin- sensitive and -insensitive mechanisms of the contractile ac- tion of P on the intestine. The current isolated organ study in the guinea-pig ileum, urinary bladder and trachea (a) con- firms the presence of such components of effect (ileum and bladder); (b) indicates TRPV1 involvement in the effect of 5 or 30 μmol/l of P on the basis of an inhibitory action of the antagonist BCTC (ileum); (c) indicates that HC 030031-sensi- tive TRPA1 receptors and nifedipine-sensitive Ca2+ channels contribute to the capsaicin-resistant contraction to 30 μmol/l P (ileum) and (d) shows that the contractile effect of P up to 100 μmol/l (guinea-pig trachea) or 30 μmol/l (guinea-pig uri- nary bladder) is capsaicin-sensitive and mediated by TRPV1 receptors/channels.

Introduction

Piperine (P), a pungent substance found in black pep- per, is a sensory stimulant agonist on TRPV1 receptors/ channels, structures also sensitive to noxious heat and capsaicin (the hot substance in red peppers) (cf. [1–4]). The mechanism of action of P on the isolated guinea-pig small intestine seems only partly identical with that of cap- saicin [5]. High concentrations of capsaicin can render a subset of sensory nerve endings inactive for a long time, that is, practically for the whole duration of an in vitro ex- periment (in vitro desensitization) [2, 6, 7]. Both capsaicin and P cause the ileum to contract but, as opposed to cap- saicin (whose effect is fully absent following capsaicin de- sensitization), the contractile action of P is capsaicin-sen- sitive at low concentrations only, while higher P concen- trations retain their effectiveness after capsaicin desensitization [5]. TTX plus substance P desensitization have been reported to strongly inhibit the effect of a low, but only weakly that of a high, concentration of P [5]. These findings led to the conclusion that P has a non-spe- cific contractile effect in the ileum. Given the progress made in the field of TRP pharmacology (e.g. the appear- ance of specific antagonists), as well as a possible TRPA1 agonist effect of P [4], we have planned experiments to get closer to the mechanism of the action of P, especially in the higher concentration range, in the small intestine and other isolated organs of the guinea-pig.

Materials and Methods

The experiments have been approved by the Regional Commit- tee for Animal Research, as well as by the respective committee of University of Pécs. These experiments were of the ‘ex-vivo’ type and are in accordance with the European Guidelines on Labora- tory Animal Care. Guinea-pigs (short-haired, colored) of either sex weighing 300–500 g were stunned by a blow to the occiput and bled out. Whole segments of ileum, approximately 2 cm in length, were cleared of contents and set up as preparations. The terminal 5 cm of the small intestine was not used. Preparations were sus- pended in organ baths containing 5 ml of oxygenated (95% O2, 5% CO2) Krebs-Henseleit solution at 37 °C. Composition of the bath- ing solution was as follows (mmol/l), NaCl 119, NaHCO3 25, KCl 2.5, MgSO4 1.5, CaCl2 2.5, KH2PO4 1.2 and glucose 11. Movements of the tissues were recorded isotonically, by means of lever trans- ducers and bridge amplifiers (Hugo Sachs – Harvard Apparatus, March-Hugstetten, Germany). The load on the tissues was 7 mN. Experiments commenced after an equilibration period of 40 min, whereafter maximal contraction was evoked with histamine (3 μmol/l).

Fig. 1. Original tracings showing the con- tractile response to P (30 μmol/l, added at arrowheads) of the guinea-pig whole ile- um, urinary bladder strip or the tracheal ‘zigzag’ preparation. Vertical bars indicate the maximal spasm of the preparation evoked by histamine (ileum) or KCl (blad- der and trachea). Horizontal bars: 2 min.

Guinea-pig tracheal ‘zigzag’ and bladder strip preparations were made as described earlier [8]; for both tissues, 2 preparations were made from 1 animal. The load on the tissues was 3 and 5 mN, respectively. Maximal contraction was evoked with KCl (80 mmol/l) at the end of the experiments.
Drugs used were atropine sulfate, capsaicin, histamine dihydro- chloride, pyridoxalphosphate-6-azophenyl-2’,4’-disulphonic acid (PPADS, P2 purinoceptor antagonist), tetrodotoxin (TTX; Sigma), 4-(3-chloro-2-pyridinyl)-N-(4-(1,1-dimethylethyl)phenyl)-1-pi- perazinecarboxamide (BCTC; TRPV1/TRPM8 antagonist), α,β- methylene ATP, methysergide, nifedipine (1-butyl-4-piperidinyl) methyl-8-amino-7-chloro-1,4-benzodioxane-5-carboxylate (SB 204 070), tropisetron, 1,2,3,6-tetrahydro-1,3-dimethyl-N-(4-(1- methylethyl)phenyl)-2,6-dioxo-7H-purine-7-acetamide, 2-(1,3-Dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)- N-(4-isopropylphenyl)acetamide (HC 030031, TRPA1 antagonist; Tocris) and chloropyramine hydrochloride (Egis Pharma, Hungary). Capsaicin desensitization was performed by a 10-min exposure to a high concentration of the drug (10 μmol/l), which was followed by a 60-min washout period, except in the trachea, where the washout period was extended to 120 min. For inhibiting the ef- fect of serotonin, we used a combination of receptor antagonists (methysergide, 0.3 μmol/l, predominantly inhibiting 5-HT2 recep- tors; tropisetron, 3 μmol/l, for 5-HT3 receptors and SB 204070, 3 μmol/l, for 5-HT4 receptors). Preliminary experiments had shown that this combination fully inhibited the contractile effect of sero- tonin (0.1–3 μmol/l), without reducing cholinergic ‘twitch’ re- sponses to electrical field stimulation.

Results

Guinea-Pig Ileum

P (1, 3, 10, 30 and 100 μmol/l) caused concentration-dependent contractions (average, 1.0, 12, 38, 72 and 69.5% of maximum, respectively; n = 5–10). The response fully developed within 30 s, at least partially faded within the 5-min period of observation (fig. 1) and showed limited reproducibility after 45 min. Hence, independent groups of preparations have been tested with only one administra- tion of P. We selected 5 μmol/l as the smaller and 30 μmol/l as the higher concentration to be analyzed. The effect of the smaller concentration was strongly inhibited by capsa- icin desensitization, the TRPV1/TRPM8 receptor antago- nist BCTC, TTX and atropine (64, 54, 78 and 63% reduc- tion, respectively; table 1). The histamine H1 receptor an- tagonist chloropyramine, the P2 purinoceptor antagonist PPADS (n = 4 for both) and the TRPA1 receptor/channel antagonist HC 030031 (table 1) failed to significantly affect the response to P (5 μmol/l). P (30 μmol/l) elicited a high- er response, which was inhibited by the same drugs, but to a smaller extent. Reductions were 29% with capsaicin de- sensitization, 53% with BCTC, 39% with TTX and 45% with atropine (table 1). Chloropyramine or a combination of serotonin 5-HT1, 5-HT2 and 5-HT3 receptor antago- nists (see ‘Materials and Methods’) failed to influence the response to 30 μmol/l P (n = 4 and 6, respectively; data not shown).

Guinea-pig ileum

Another protocol was used for studying the direct con- tractile effect of P (30 μmol/l) on the smooth muscle. These preparations were subjected to capsaicin desensiti- zation and TTX treatment. Under these circumstances, atropine or HC 030031 significantly inhibited the re- sponse (table 1), but BCTC was ineffective (table 1). Not surprisingly, the Ca2+-channel blocker nifedipine fully.

Guinea-Pig Bladder and Trachea

In the bladder, even a high P concentration (30 μmol/l) evoked responses (fig. 1) that were strongly inhibited by capsaicin desensitization or BCTC (table 1). TTX or a combination of α,β-methylene ATP desensitization (10 + 10 μmol/l) and atropine (inhibitors of the 2 main compo- nents of parasympathetic functional innervation) were ineffective (n = 4). At a still higher P concentration (100 μmol/l), the responses were smaller than at 30 μmol/l; they were not significantly influenced by capsaicin desen- sitization, but prevented by nifedipine pretreatment. P (3 μmol/l) was practically ineffective.
Discussion

Several lines of evidence suggest that P is an agonist at TRPV1 receptors/channels [3, 4, 9, 10]. These receptors are present in high density at capsaicin-sensitive sensory nerve endings that can be functionally blocked by capsa- icin desensitization (see among others [2]).Previous results on the guinea-pig trachea showed that the contractile effect of P (up to 35 μmol/l) is capsaicin sensitive [11]. We now confirmed and extended this find- ing; the inhibitory action of BCTC on the effect of P indi- cates TRPV1 involvement. Similar to the trachea, the guinea-pig bladder is a preparation in which capsaicin causes contraction that is sensitive to previous capsaicin desensitization, but does not involve TTX-sensitive mechanisms [2, 12]. It is assumed that sensory neu- rotransmitters (first of all tachykinins) are released from sensory nerve endings in a TTX-resistant fashion (most probably by stimulating TRPV1) and act on the smooth muscle directly in these 2 preparations; hence, no intrinsic neurons are involved in the capsaicin-induced con- traction. In the bladder, a moderate capsaicin-resistant contraction was evoked by 100 μmol/l of P.

In the guinea-pig small intestine, capsaicin has been shown to act in a more complicated way; sensory neurons seem to activate intrinsic (predominantly cholinergic) nerves and the contractile response is largely cholinergic in nature [2, 6, 7]. Takaki et al. [5] have shown that P has capsaicin-like effects in a small concentration (1 μmol/l), but causes a mainly capsaicin-resistant contraction at a 10-fold higher concentration. While we essentially con- firmed these findings in the current work, our primary aim was to get more insight into the capsaicin-resistant contractile effect of P, with special regard to the direct ex- citation of smooth muscle. The effect of P (30 μmol/l) is not mediated by histamine or serotonin, given the inef- fectiveness of the respective antagonists.

On capsaicin-desensitized, TTX-treated preparations (i.e. in which capsaicin-sensitive sensory neurons, as well as axonal conduction in most types of neurons, were blocked) P still has an excitatory effect, inhibited by the Ca2+-channel blocker nifedipine. The 2 findings that came to us as a surprise in this arrangement were the in- hibitory effects of HC 030031 and of atropine. The TRPA1 antagonist does not reduce either the cholinergic ‘twitch’ response to electrical field stimulation of nerves or the ef- fect of exogenous acetylcholine (unpublished observa- tions from our laboratory). This indicates that a non-spe- cific inhibition of the smooth muscles or nerves is im- probable. Hence, we assume that TRPA1 receptors may contribute to the muscle-contracting effect of a high concentration of P. Without capsaicin + TTX pretreatment, capsaicin-sensitive and other mechanisms probably mask the TRPA1-mediated effect (although a statistically non- significant inhibitory effect of HC 030031 may appear to be present; table 1). Another conclusion that can be drawn is that the effect(s) of P in this arrangement take place on structures that are different from capsaicin-sensitive nerves and TTX-sensitive tissue elements. The inhibitory action of atropine (in spite of TTX treatment) may reflect a spontaneous ‘spill’ of acetylcholine from cholinergic nerve endings in the absence of action potentials or cho- linergic neurons firing TTX-resistant spikes. The ineffec- tiveness of BCTC may indicate that not only TRPV1 but also TRPM8 receptors/channels are not involved [1].In conclusion, these data indicate that P-sensitive structures (including TRPA1) are partly located outside the capsaicin-sensitive nerves in the guinea-pig ileum and contribute to the excitatory action of this drug.

Acknowledgments

This study was supported by a postdoctoral fellowship of the Medical School, University of Pécs. The Department of Pharma- cology and Pharmacotherapy, University of Pécs, Hungary (Prof. E. Pinter), also provided help in many different ways. The authors express their sincere thanks for the support.

Disclosure Statement

The authors declare no conflict of interest.

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