Figure 6. Potentiation of ICC electrical activity with 5-HT (10 mM) and 2-Me-5-HT (10 mM). All measurements were carried out in the presence of nifedipine (1 mM) and TTX (250 nM). doi:10.1371/journal.pone.0024928.g006 Ca2+ pathway may simultaneously contribute to the enhancement of ICC [Ca2+]i activity. Five subunits (i.e. 5-HT3A–E) are known to form the 5-HT3 receptor complex, and changes in the composition alter Ca2+- permeability of this channel [46]. In future studies, it would be of interest to elucidate the composition of 5-HT3A-E receptor subtypes and how 5-HT3 receptors are coupled to Ca2+ release channels in intracellular Ca2+ stores to generate pacemaker [Ca2+]i activity. Polymorphism of these 5-HT3 receptor subunit genes seems likely to affect gut motility by modulating ICC as well as neuronal activities [12], and underlies some functional disorders [47]. Furthermore, RT-PCR examinations detected transcripts of other 5-HT receptor genes in ICC i.e. 5-HT2 and 5-HT4 receptor genes [48,49]. Recent studies have shown that 5-HT2B receptor antagonists reduced proliferation of cultured ICC, and that the small intestine of mice lacking 5-HT2B receptors contains less ICC in the myenteric and deep muscular plexuses, although intestinal transit is not significantly slowed [50,51]. On the other hand, 5- HT4 receptor antagonists impair the regeneration of enteric neurons after surgical operation and their development in gut-like organs derived from mouse embryonic stem cells, with indistin- guishable changes in the ICC network [21,52]. It is likely that 5- HT causes numerous effects via these different 5-HT receptors, depending on cell type, location of the gut, and the stage of development and aging. The scenario for 5-HT augmentation of ICC activity is possibly modified by the roles of adjacent cells in the actual gut. In the present study, we applied nifedipine and TTX to clearly demonstrate the effect of 5-HT on ICC; however, smooth muscle cells and enteric neurones suppressed by these inhibitors may also be involved, because coordinated actions of these cells produce gut motility [4]. For example, as seen in Fig. 5, ICC pacemaker activity propagates on the luminal plane. Indeed, electric conduction of gut pacemaker activity along the musculature can be detected magnetically [53]. It is thought that ICC and smooth muscle cells are electrically connected [54]. Therefore, under normal conditions (without DHP Ca2+ antagonists), in addition to network-forming processes in ICC, the smooth muscle bundle conducts a part of electric current generated by a group of ICC, Serotonin Augments Gut Pacemaker Activity and amplifies pacemaker activity in adjacent ICC, because it is thought that ICC possess a mechanism to transform depolarisation in the plasma membrane into activation of [Ca2+]i oscillations for pacemaking [55,56]. Also, some populations of enteric neurones may release activators for ICC pacemaker activity in response to 5-HT. In the myenteric plexus, serotonergic neurones are involved in descending contraction [9,10], and it is known that ICC express numerous receptors for excitatory neurotransmission, e.g. pur- inoceptors, neurokinin and acetylcholine receptors [57–60]. These molecules may access ICC to activate in parallel. The present finding on augmentation of ICC activity via 5-HT3 receptors implies pharmacological interventions on gut motility disorders. For example, irritable bowel syndrome, classified into two types, i.e. diarrhoea- and constipation-dominant IBS (d-IBS and c-IBS), is known to involve 5-HT-related mechanisms along with infectious and inflammatory changes. Excess 5-HT due to impairment of reuptake transport is ascribed to some populations of d-IBS [61–63]. Also, antineoplastic drugs, e.g. cisplatinum, stimulate 5-HT release [64–66]. In such cases, it is rational to suppress ICC pacemaker activity in addition to nervous activities by blocking 5-HT3 receptors. It is speculated that stimulation of 5- HT3 receptors in enteric neurons and ICC synergically facilitates gut contractility and afferent neural activity toward the brain. Thereby, 5-HT3 receptors in the gut may contribute to the gut- brain axis. As seen in murine ileal ICC, we have also observed that 5b-oHthTC3 ar2e+caenpdtoerleacgtroincipstascepmotaeknetriaatcet,ivwithieisleinatnhteagmounrisintse ssutopmpraecshs in preliminary experiments. Although extensive studies are required in model animals and humans, 5-HT is likely to enhance ICC pacemaker activity throughout the gastrointestinal tract. Similar regulatory mechanisms may underlie other peripheral spontaneous activities. Evidence is being accumulated that ICC- like interstitial cells ubiquitously exist in many organs and tissues that are effectors of the autonomic nervous system, such as the ureter, urinary bladder, urethra, uterus, lymph ducts, veins, etc, suggesting their possible contribution to spontaneous activity [67– 71]. Also, in some ICC-like cells, spontaneous [Ca2+]i and electric activities have already been demonstrated. In the light of regulatory mechanisms of ICC and ICC-like cells, investigating functional disorders related to a wide range of peripheral spontaneous rhythmicity, e.g. irritable bladder, is merited. In summary, the present study has shown 5-HT augmentation of ICC pacemaker activity via 5-HT3 receptors. Since 5-HT3 receptors are Ca2+-permeable nonselective cation channels, this effect on ICC activity is presumably through enhancement of Ca2+ influx from the extracellular space, through itself and simultaneous activation of voltage-gated Ca2+-permeable channels. ICC appear to be a promising target in functional motility disorders in the gut. Supporting Information Figure S1 An example of 5-HT-augmentation of ICC pace- maker [Ca2+]i oscillations in an ileal musculature preparation. Ileal musculature segments (,5 mm620 mm) containing the myenter- ic plexus, the same preparation used in 868 MEA measurements, were loaded with Fluo-3AM. Fluo-3 emission light images were continuously monitored the same as cell cluster experiments. The extracellular solution contained 1 mM nifedipine and 250 nM TTX. A) A control fluorescent image. B and C) Series of ratio images of a [Ca2+]i oscillation cycle in control and in the presence of 5-HT (10 mM), respectively. Each image was acquired at 300 ms intervals. Video S1 and Video S2 correspond to ratio images shown in B and C, respectively. Note that green represents the Ft/F0 ratio of ,1 in cell cluster preparations (Fig. 1A) to PLoS ONE | www.plosone.org 8 September 2011 | Volume 6 | Issue 9 | e24928