Effects of menthol on circular smooth muscle of human colon: Analysis of the mechanism of action

Abstract

Menthol is the major constituent of peppermint oil, an herbal preparation commonly used to treat nausea, spasms during colonoscopy and irritable bowel disease. The mechanism responsible for its spasmolytic action remains unclear. The aims of this study were to investigate the effects induced by menthol on the human distal colon mechanical activity in vitro and to analyze the mechanism of action. The spontaneous or evoked-contractions of the circular smooth muscle were recorded using vertical organ bath. Menthol (0.1 mM–30 mM) reduced, in a concentration-dependent manner, the amplitude of the spontaneous contractions without affecting the frequency and the resting basal tone. The inhibitory effect was not affected by 5-benzyloxytryptamine (1 μM), a transient receptor potential-melastatin8 channel antagonist, or tetrodotoxin (1 μM), a neural blocker, or 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (10 µM), inhibitor of nitric oxide (NO)-sensitive soluble guanylyl cyclase, or tetraethylammonium (10 mM), a blocker of potassium (K⁺)-channels. On the contrary, nifedipine (3 nM), a voltage-activated L-type Ca²⁺ channel blocker, significantly reduced the inhibitory menthol actions. Menthol also reduced in a concentration-dependent manner the contractile responses caused by exogenous application of Ca²⁺ (75–375 μM) in a Ca²⁺-free solution, or induced by potassium chloride (KCl; 40 mM). Moreover menthol (1–3 mM) strongly reduced the electrical field stimulation (EFS)-evoked atropine-sensitive contractions and the carbachol-contractile responses. The present results suggest that menthol induces spasmolytic effects in human colon circular muscle inhibiting directly the gastrointestinal smooth muscle contractility, through the block of Ca²⁺ influx through sarcolemma L-type Ca²⁺ channels.

Introduction

Menthol is a cyclic monoterpene alcohol which is found as a major constituent of peppermint (Mentha piperita L) oil, an herbal preparation commonly used in traditional medicine to treat various gastrointestinal disorders, such as nausea and vomiting (Haniadka et al., 2012), pain caused by endoscopy (Asao et al., 2001) and irritable bowel disease (Ford et al., 2008). In vitro and in vivo animal studies have documented the inhibitory effects of menthol on gastrointestinal motility (Hawthorn et al., 1988, Grigoleit and Grigoleit, 2005, Amato et al., 2013), justifying its spasmolytic effects. However, previous studies about the effect of peppermint oil on the human colonic contractility seem to be contradictory. Using similar techniques and doses of peppermint oil, some workers have shown decreased motility (Duthie, 1981) whereas others report increased colonic contraction with spasm (Rogers et al., 1988).

To date, the site of action and pathways mediating the effects of peppermint oil and other menthol-containing products remain controversial (Hawthorn et al., 1988, Hills and Aaronson, 1991, Mahmood et al., 2003, de Araújo et al., 2011). Despite the Ca²⁺-channel antagonism seems to be the most likely pharmacological effect (Grigoleit and Grigoleit, 2005), it may not to be the unique mechanism of action underlying the antispasmodic effect of peppermint oil in intestinal tract. In fact, the gastrointestinal effect of menthol could be, at least in part, mediated by the transient receptor potential-melastatin8 (TRPM8) channel, a nonselective cation channel, gated by cold stimuli and menthol. The activation of TRPM8 by menthol results in an increase in intracellular Ca²⁺ concentration (Peier et al., 2002, Reid et al., 2002) and menthol-induced release of Ca²⁺ from intracellular stores enhances neurotransmission at sensory sinapses (Tsuzuki et al., 2004). The TRPM8 channels are widely expressed in sensory neurons in dorsal root and trigeminal ganglia (McKemy et al., 2002). Concerning the gastrointestinal tract, TRPM8 channels have been localized in rat gastric and colonic muscle (Mustafa and Oriowo, 2005), in mouse small and large intestine mucosa and myenteric plexus (Zhang et al., 2004, Ramachandran et al., 2013) and in human colon (Ramachandran et al., 2013). Moreover, menthol has been reported to be able to modulate other membrane proteins such as TRP-ankyrin 1 (TRPA1) ion channels (Karashima et al., 2007), mainly expressed in enteric neurons (Zhang et al., 2004), 5-hydroxytryptamine 3 receptors (Ashoor et al., 2013) and α7-nicotine receptors (Kabbani, 2013). Menthol is also able to block the voltage-gated neuronal and skeletal muscle Na⁺-channels in resting and inactivated states (Haeseler et al., 2002).

Therefore, the aims of the present study were (a) to assess the effects of menthol on the mechanical activity of human distal colon circular smooth muscle in vitro and (b) to characterize the mechanism of action involved in the responses observed.