Noncompetitive inhibition by camphor of nicotinic acetylcholine receptors

doi:10.1016/S0006-2952(01)00547-0

Biochemical Pharmacology

Volume 61, Issue 7, 1 April 2001, Pages 787-793

https://doi.org/10.1016/S0006-2952(01)00547-0 Get rights and content

Abstract

The effect of camphor, a monoterpenoid, on catecholamine secretion was investigated in bovine adrenal chromaffin cells. Camphor inhibited [³H]norepinephrine ([³H]NE) secretion induced by a nicotinic acetylcholine receptor (nAChR) agonist, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), with a half-maximal inhibitory concentration (ic₅₀) of 70 ± 12 μM. In addition, camphor inhibited the rise in cytosolic calcium ([Ca²⁺]_i) and sodium ([Na⁺]_i) induced by DMPP with ic₅₀ values of 88 ± 32 and 19 ± 2 μM, respectively, suggesting that the activity of nAChRs is also inhibited by camphor. On the other hand, binding of [³H]nicotine to nAChRs was not affected by camphor. [Ca²⁺]_i increases induced by high K⁺, veratridine, and bradykinin were not affected by camphor. The data suggest that camphor specifically inhibits catecholamine secretion by blocking nAChRs without affecting agonist binding.

Introduction

²Camphor, one of the naturally occurring monoterpenoids, is a volatile and aromatic compound. It is a major component of Cinnamomum camphora, which is found in temperate regions such as Java, Sumatra, China (central provinces), Korea, Japan, Formasa, and Brazil. The essential oil is also present in Piper angustifolium[1], Sassaafras albidum[2]Jasonia candicans, and J. montana[3]. Camphor has been widely used as a fragrance in cosmetics, as a flavouring food additive, as a scenting agent in a variety of household products, as an active ingredient in some old drugs, and as an intermediate in the synthesis of perfume chemicals [4]. In addition, the monoterpenoid is present in a number of over-the-counter medications, mainly for external application, and is readily available in drugstores [5]. Camphor exhibits a broad range of biological properties. It has been used as an insect repellent [6], a bacteriostatic and fungistatic agent [1], [2], [3], and an antitussive [7]. On the other hand, the effect of camphor on cellular signal transduction has received little attention. We investigated the effects of camphor on catecholamine secretion and calcium increase induced by various stimulants and found that camphor specifically inhibits nAChRs, thereby blocking nAChR-mediated calcium and sodium increases and catecholamine secretion.

Section snippets

Materials

Camphor, DMPP, bradykinin, and veratridine were purchased from the Sigma Chemical Co. Fura-2/AM, SBFI/AM, and Pluronic F-127 were purchased from the Molecular Probes, Inc. [³H]NE and [³H]nicotine were purchased from the NEN Life Science Products.

Chromaffin cell preparation

Chromaffin cells were isolated from bovine adrenal medulla by a two-step collagenase digestion as previously described [8]. For the measurement of [³H]NE secretion and the [³H]nicotine binding assay, cells were plated in 24-well plates at a density of 5

Inhibition by camphor of DMPP-induced [³H]NE secretion

To study the effects of camphor on catecholamine secretion, we treated [³H]NE-loaded chromaffin cells with camphor. Whereas camphor (up to 300 μM) by itself did not induce [³H]NE secretion (data not shown), it decreased the DMPP-induced secretion of [³H]NE in a concentration-dependent manner with a half-maximal inhibitory concentration (ic₅₀) of 70 ± 12 μM (Fig. 1). Camphor at 300 μM completely blocked the DMPP-induced secretion of [³H]NE.

Inhibition by camphor of the DMPP-induced rise in [Ca²⁺]_i

Since an increase in [Ca²⁺]_i is an essential step in

Discussion

Our data clearly indicate that camphor specifically inhibits nAChRs, thereby causing inhibition of catecholamine secretion. Since all tested responses induced by DMPP including [³H]NE secretion and [Ca²⁺]_i and [Na⁺]_i increases were inhibited by camphor, it is highly plausible that the activity of nAChRs was inhibited by the reagent. On the other hand, [Ca²⁺]_i increases induced by high K⁺, veratridine, and bradykinin were not affected by camphor, which suggests that camphor has no effect on

Acknowledgements

We thank MyoungSin Company, Inc., especially Mr. Sang-Yul Kim, chief director, and Mr.Jun-Hun Kim, for providing the bovine adrenal glands. This research was supported by the Brain Science and Engineering Research Program and the National Research Laboratory Program sponsored by the Korean Ministry of Science and Technology. This work was also supported by the Brain Korea 21 Program of the Ministry of Education and by a grant from GenoMine Inc.

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This modulatory effect of menthol is similar to our observed effects on the levamisole sensitive receptor. Monoterpenoids are a group of plant secondary metabolites that have been shown to modulate the function of nicotinic acetylcholine receptors of mammals and insects (Lozon et al., 2016; Park et al., 2001, 2003; Tong et al., 2013). We were interested in finding possible agonists, antagonists or modulators for these cys loop receptors of parasitic nematodes.
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In recent decades, reproductive problems were raised alongside the continuous genetic selection for performance purposes in poultry. As such, a negative correlation was observed between reproductive and production performance, which may possibly imply that the high growth rate in poultry causes physiological disorders such as decreased libido and frequency of mating. A study was conducted to establish the efficacy of feeding graded levels of camphor on reproductive function of Japanese quail as a bird model. Two-hundred-and-eighty one-day-old quail chicks were randomly assigned to individual cages in 7 treatments (zero [control], 250, 500, 750, 1,000, 5,000, or 10,000 ppm of camphor). The results demonstrated that testosterone concentration displayed an uncertain attitude, as the lower dosages of camphor used (250, 500, and 750 ppm) had the highest values. Thyroxine (T₄) linearly increased with increasing camphor levels in male birds (P < 0.05). Triiodothyronine (T₃) also was increased in the groups receiving a camphor-containing diet in comparison with the control (P < 0.05). The male birds’ libido behavior was linearly increased with the increase of camphor concentration (P < 0.05). Dietary camphor in 500 and 10,000 ppm caused the highest percentage of fertility (P < 0.0001) and hatchability (P = 0.0117), respectively. As well, the groups with 5,000 and 10,000 ppm of camphor accounted for the highest percentage of hatched-fertile eggs (P = 0.0055). The highest rate of sperm penetration in the yolk inner perivitelline layer (IPVL) was demonstrated by the group allotted with 10,000 ppm camphor (P < 0.05). This group also had the highest hole persistency in IPVL. Conclusively, the result indicated that camphor can be added in poultry diet for the improvement of reproductive functions.
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It is possible that carvacrol is able to inhibit more strongly the altered AChE in organophosphate-resistant SCFT than the normal AChE of susceptible ticks accounting for the resistance ratio of 3.2 observered in this study. Previous studies on monoterpenoid mode of action have revealed several possible biochemical targets in the nervous system of vertebrates and invertebrates, including ionotropic γ–aminobutyric acid (GABA) receptors (Priestley et al., 2003; Tong and Coats, 2010; Hold et al., 2000; Tong and Coats, 2012), octopamine receptors (Enan, 2001; Enan, 2005a), tyramine receptors (Enan, 2005b; Lei and Enan, 2010), nicotinic acetylcholine receptors (Park et al., 2001, 2003), thermotransient receptors (Macpherson et al., 2006; Parnas et al., 2009), and acetylcholinesterase (Miyazawa et al., 1997). However, the results of correlation between toxicity for insects and acetylcholinesterase inhibition for the monoterpenes are contradictory (Ryan and Byrne, 1988; Lee et al., 2001a,b).
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¹: T-J. Park and H-K. Seo contributed equally to this work.

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Noncompetitive inhibition by camphor of nicotinic acetylcholine receptors

Abstract

Introduction

Section snippets

Materials

Chromaffin cell preparation

Inhibition by camphor of DMPP-induced [3H]NE secretion

Inhibition by camphor of the DMPP-induced rise in [Ca2+]i

Discussion

Acknowledgements

Mutat Res

Forensic Sci Int

Pulm Pharmacol

Mol Cells

Brain Res

J Biol Chem

J Physiol (Paris)

Eur J Pharmacol

J Biol Chem

Eur J Pharmacol

Chemical composition and antimicrobial activity of essential oil of Piper angustifolium

Planta Med

Chemical composition of essential oil from the root bark of Sassafras albidum

Planta Med

Chemical composition and antimicrobial activity of essential oils of Jasonia candicans and J. montana

Planta Med

Relative efficacy of various oils in repelling mosquitoes

Indian J Malariol

Inhibition by camphor of DMPP-induced [³H]NE secretion

Inhibition by camphor of the DMPP-induced rise in [Ca²⁺]_i