Pulmonary, gastrointestinal and urogenital pharmacology
Expression and motor functional roles of voltage-dependent type 7 K+ channels in the human taenia coli

https://doi.org/10.1016/j.ejphar.2013.09.061Get rights and content

Abstract

Voltage-dependent type 7 K+ (KV7 or KCNQ) channels modulate the excitability of neurons and muscle cells. The aims of the present study were to investigate the motor effects of KV7 channel modulators and the expression of KV7 channels in the human taenia coli. The effects of KV7 channel modulators on the muscle tone of human taenia coli strips were investigated under nonadrenergic non-nitrergic conditions by organ bath studies. Gene expression and tissue localisation of channels were studied by real-time PCR and immunohistochemistry, respectively. Under basal conditions, the KV7 channel blocker XE-991 induced concentration-dependent contractions, with mean EC50 and Emax of 18.7 μM and 30.5% respectively of the maximal bethanechol-induced contraction, respectively. The KV7 channel activators retigabine and flupirtine concentration-dependently relaxed the taenia coli, with mean EC50s of 19.2 μM and 29.9 μM, respectively. Retigabine also relaxed bethanechol-precontracted strips, with maximal relaxations of 79.2% of the bethanecol-induced precontraction. The motor effects induced by the KV7 channel modulators were not affected by tetrodotoxin or ω-conotoxin GVIA. XE-991 greatly reduced retigabine- and flupirtine-induced relaxations. Transcripts encoded by all KCNQ genes were detected in the taenia coli, with KCNQ4 showing the highest expression levels. KV7.4 channels were clearly visualised by immunohistochemistry in colonic epithelium, circular muscle layer and taenia coli. KV7 channels appear to contribute to the resting muscle tone of the human taenia coli. In addition, KV7 channel activators significantly relax the taenia coli. Thus, they could be useful therapeutic relaxant agents for colonic motor disorders.

Introduction

The contractile activity of the gastrointestinal smooth muscle is regulated by ion channels located in the plasma membrane of the syncytium formed by smooth muscle cells, interstitial cells of Cajal and fibroblast-like cells (Sanders et al., 2012). Such contractile activity is triggered by the increase in cytosolic Ca2+ concentration, mainly determined by Ca2+ influx through voltage-gated Ca2+ (CaV1.2) channels (Sanders et al., 2012). The open probability of CaV1.2 channels is modulated positively by non-selective cation channels and negatively by K+ channels (Sanders et al., 2012). The latter play important roles in determining the resting membrane potential and modulating cell excitability. K+ channel subtype diversity contributes to the variability of membrane resting potential of the cells composing the syncytium, and so to the different levels of spontaneous tone observed in the various gastrointestinal segments (Sanders, 2008). In addition, K+ channels open in response to various stimuli, including receptor activation by neurotransmitters, hormones and paracrine substances, thus hyperpolarizing the plasma membrane and relaxing the smooth muscle cells or reducing their excitability (Sanders, 2008).

Many K+ channels are expressed in the colonic syncytium. Voltage-dependent K+ (KV) channels, in particular KV1.2, 1.5, 2.2, 4, 11 and 7, are among the most important K+ channels that regulate colonic smooth muscle contractility (Koh et al., 2012). KV7 channels are encoded by the KCNQ genes and include 5 subtypes (KV7.1–7.5). They play important roles in regulating the membrane potential of various cell types, including cardiomyocytes, neurons and smooth muscle cells (Soldovieri et al., 2011), and are abundantly expressed in vascular, tracheobronchial, genitourinary and gastrointestinal tissues, where they regulate smooth muscle contractility (Greenwood and Ohya, 2009, Jepps et al., 2009, McCallum et al., 2009, McCallum et al., 2011, Rode et al., 2010, Ipavec et al., 2011, Brueggemann et al., 2012). As for the gastrointestinal tract, KV7 channel expression has been shown in mouse stomach, jejunum and colon (Jepps et al., 2009), and rat stomach (Ohya et al., 2002, Ipavec et al., 2011). In all these segments, KV7.4 and 7.5 channel gene transcripts show the highest levels in muscular layers (Jepps et al., 2009, Ipavec et al., 2011). In addition, KV7 channel blockers and activators induce excitatory and inhibitory motor effects, respectively, in the rat gastric fundus (Ipavec et al., 2011) and in the mouse distal colon (Jepps et al., 2009).

In this study, we investigated the effects of KV7 channel modulators on the contractile activity of the human taenia coli. We also studied KV7 channel transcript and protein expression in the human taenia coli by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry, respectively. The results of the present study indicate that KV7 channels contribute to the regulation of the basal muscle tone of the human taenia coli. In addition, the activation of KV7 channel produces profound relaxant effects in the human taenia coli, suggesting that these channels could be novel pharmacological targets for therapeutic interventions against colonic motor disturbances.

Section snippets

Policy and ethics

This study was approved by the institutional Ethical Committee of the Catholic University. All patients signed an informed consent.

General methods

Colonic segments, 3–4 cm long, were obtained from patients undergoing surgical resections for colonic cancer. Each colon segment was cut at one margin of the surgical specimen immediately after resection and appeared macroscopically normal. The new margin that was created on the surgical specimen after removal of the segment was always assessed by pathologists and in

Motor effects of XE-991 compared to those of bethanechol

The muscarinic agonist bethanechol (0.1–100 μM) significantly contracted the human taenia coli strips, with an EC50 of 3.26±0.51 μM (n=8; Fig. 1A). Similarly, the KV7 blocker XE-991 (1–100 μM) also produced concentration-dependent contractions of the taenia coli (Fig. 1B), although it showed lower potency and efficacy; in fact, the mean EC50 and Emax of XE-991-induced taenia coli contraction were 18.7±3.7 μM and 30.5±4.9% (n=9, four transverse, three descending, one ascending and one sigmoid colon

Discussion

Many K+ channels, including the slowly delayed-rectifiers encoded by the KCNQ (KV7) gene family, are functionally important in the colon (Koh et al., 2012). KV7 channels are actually more known for their involvement in the physiology of the vascular smooth muscle than in that of gastrointestinal smooth muscle. The first studies in the smooth muscle field reported that the KV7 channel blockers linopirdine and XE-991 inhibited a sustained outwardly rectifying K+ current, depolarised the resting

Acknowledgements

Supported by Fondi Ateneo of the Catholic University of the Sacred Heart, Rome. The technical help of Dr. P. Lanza (Institute of Histopathology, Catholic University of the Sacred Heart, Rome) with the immunohistochemistry experiments is deeply acknowledged.

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