Serotonin Augments Gut Pacemaker Activity via 5-HT3 Receptors Hong-Nian Liu1, Susumu Ohya3, Yuji Nishizawa 2 , Kenta Sawamura1, Satoshi Iino 4 , Mohsin Md Syed1¤, Kazunori Goto1, Yuji Imaizumi3, Shinsuke Nakayama 1 * 1 Department of Cell Physiology, Nagoya University Graduate School of Medicine, Nagoya, Japan, 2 Department of Anatomy and Cell Biology, Nagoya University Graduate School of Medicine, Nagoya, Japan, 3 Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya, Japan, 4 Department of Anatomy, Faculty of Medical Sciences, University of Fukui, Fukui, Japan Abstract Serotonin (5-hydroxytryptamine: 5-HT) affects numerous functions in the gut, such as secretion, muscle contraction, and enteric nervous activity, and therefore to clarify details of 5-HT’s actions leads to good therapeutic strategies for gut functional disorders. The role of interstitial cells of Cajal (ICC), as pacemaker cells, has been recognised relatively recently. We thus investigated 5-HT actions on ICC pacemaker activity. Muscle preparations with myenteric plexus were isolated from the murine ileum. Spatio-temporal measurements of intracellular Ca2+ and electric activities in ICC were performed by employing fluorescent Ca2+ imaging and microelectrode array (MEA) systems, respectively. Dihydropyridine (DHP) Ca2+ antagonists and tetrodotoxin (TTX) were applied to suppress smooth muscle and nerve activities, respectively. 5-HT significantly enhanced spontaneous Ca2+ oscillations that are considered to underlie electric pacemaker activity in ICC. LY- 278584, a 5-HT3 receptor antagonist suppressed spontaneous Ca2+ activity in ICC, while 2-methylserotonin (2-Me-5-HT), a 5- HT3 receptor agonist, restored it. GR113808, a selective antagonist for 5-HT4, and O-methyl-5-HT (O-Me-5-HT), a non- selective 5-HT receptor agonist lacking affinity for 5-HT3 receptors, had little effect on ICC Ca2+ activity. In MEA measurements of ICC electric activity, 5-HT and 2-Me-5-HT caused excitatory effects. RT-PCR and immunostaining confirmed expression of 5-HT3 receptors in ICC. The results indicate that 5-HT augments ICC pacemaker activity via 5-HT3 receptors. ICC appear to be a promising target for treatment of functional motility disorders of the gut, for example, irritable bowel syndrome. Citation: Liu H-N, Ohya S, Nishizawa Y, Sawamura K, Iino S, et al. (2011) Serotonin Augments Gut Pacemaker Activity via 5-HT3 Receptors. PLoS ONE 6(9): e24928. doi:10.1371/journal.pone.0024928 Editor: Markus M. Heimesaat, Charite´, Campus Benjamin Franklin, Germany Received May 18, 2011; Accepted August 19, 2011; Published September 15, 2011 Copyright: 2011 Liu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants-in-aid for scientific research from the Japan Society for the Promotion of Science, and a research grant from Japan Gut Club. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: h44673a@nucc.cc.nagoya-u.ac.jp ¤ Current address: Department of Neurobiology and Developmental Sciences, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America Introduction Special interstitial cells with abundant c-Kit receptors on their surface are distributed throughout the gastrointestinal tract. These cells are referred to as interstitial cells of Cajal (ICC) due to the histological features of the network [1–3]. It is now considered that ICC in the myenteric region act as pacemaker cells, and produce gut movements in concert with enteric neurones and smooth muscle cells [4–6]. Numerous neurotransmitters and hormones are likely to affect ICC activity, and thereby modulate gut motility. Serotonin (5-hydroxytryptamine: 5-HT), well known for mood control in the brain, also plays a crucial role in cellular signalling in the gut. Indeed, enterochromaffin cells release the majority (.90%) of 5-HT in the human body in response to the pressure of intraluminal content and other noxious stimuli [7]. Some enteric neurones in the descending peristaltic reflex pathway also release 5-HT as a neurotransmitter [8–10]. Since enteric neurones and smooth muscle cells express various 5-HT receptors depending upon cell type and location of the cell, and their functions are critically affected by this signalling molecule [11,12], 5-HT receptors are key targets in pharmacological interventions of gut functional disorders, as well as psychiatric disorders of the brain. It is thought that oscillations of the intracellular (cytosolic) Ca2+ concentration ([Ca2+]i) in ICC cells underlie gut pacemaker activity. Namely, periodic activation of Ca2+-sensitive ion channels in the plasma membrane generates pacemaker potentials [13,14]. Previously, we demonstrated that spontaneous electrical activity occurs in synchrony with [Ca2+]i oscillations in ICC, and that coordinated actions of intracellular Ca2+ release channels and transmembrane Ca2+ influx pathways underlie ICC [Ca2+]i oscillations [15–17]. In the present study, we provide evidence that 5-HT regulates ICC pacemaker activity. We performed Ca2+ imaging and potential mapping of ICC pacemaker activity using fluorescent Ca2+ probes and microelectrode array (MEA), respectively, and found that 5-HT enhances both Ca2+ and electric activities of ICC via 5-HT3 receptors, which are nonselective cation channels permeable to Ca2+. We also carried out RT-PCR and immuno- staining to confirm the expression of 5-HT3 receptors in ICC. Our findings suggest that 5-HT modulation of ICC activity should also be considered for gut motility disorders, for example, irritable PLoS ONE | www.plosone.org 1 September 2011 | Volume 6 | Issue 9 | e24928