Figure 3. RT-PCR examinations for 5-HT receptors. A) RNA samples were obtained from isolated c-KIT-immunopositive cells (ICCs) or smooth muscle cells (SMC). NTC represents ‘no template control’. RT- PCR was performed for 5-HT2B, 5-HT3A, c-kit and GAPDH (an index of proper amplification). Numbers on the right of each gel indicate the size marker (bp). B) Examples of RT-PCR detection of mast cell markers (CD68 and Cma1) in spleen (35 cycles) and c-kit-immuopositive cells (50 cycles). doi:10.1371/journal.pone.0024928.g003 oscillations in ICC persist [19]. We therefore carried out [Ca2+]i imaging in the presence of nifedipine. A typical effect of 5-HT is shown in Fig. 1A. In the continuous presence of nifedipine (1 mM), spontaneous [Ca2+]i oscillations were observed in a small limited area (near (x)) [control: Panels (a– b) in Fig. 1A]. Application of 5-HT (10 mM) enlarged the region showing spontaneous [Ca2+]i oscillations [Panels (c–d)]. The time course of changes in [Ca2+]i (Ft/F0) is plotted in Fig. 1B. In control conditions (a), only the region of interest (ROI) (x) shows [Ca2+]i oscillations with an amplitude of more than 1.2 (Ft/F0), while such Serotonin Augments Gut Pacemaker Activity activity was negligible in ROI (y) and (z). After application of 5- HT, the area yielding spontaneous [Ca2+]i oscillations enlarged, covering all three regions (x–z) synchronously. The peak amplitude in ROI (y) reached 80–90% of that in (x). On average, the area yielding [Ca2+]i oscillations increased from 11.566.0 to 42.5618.3% (n = 22), while the frequency was little affected (19.461.2 in normal vs. 19.561.7 cycles/min in the presence of 5- HT, n = 22) (Fig. 1C–E). Essentially similar effects of 5-HT were observed even in the presence of TTX as well as nifedipine (n = 5), suggesting that 5-HT augmented ICC pacemaker activity, but not through neural activity. An interesting response was observed in preparations showing intermittent [Ca2+]i oscillations. 5-HT (10 mM) caused the activity towards a continuous oscillation pattern, with a significant enlargement of the oscillation active area (n = 6) (Figure S2). When [Ca2+]i imaging was carried out in ileal cell cluster preparations from W/Wv mice, which have few pacemaking interstitial cells in this part of the small intestine (Fig. 1F–G) [27– 29], no [Ca2+]i oscillation was observed irrespective of 5-HT application (in the presence of 1 mM nifedipine, n = 4) (Fig. 1H). This result confirms that ICC are responsible for 5-HT-mediated enhancement in normal mice. 5-HT receptor agonists and antagonists Next, we assessed which receptors are responsible for 5-HT- mediated pacemaker [Ca2+]i activity. In these experiments, we used preparations showing synchronous [Ca2+]i oscillations (like the preparation shown in Fig. 1). Application of LY-278584 (10 mM), an antagonist for type 3 5-HT receptors (5-HT3) significantly decreased the amplitude of the oscillations, and the intervals between them often became irregular (middle traces in Fig. 2A–B). Almost complete recovery was achieved by additional application of 10 mM 2-methyl-5-HT (2-Me-5-HT), a selective agonist for 5-HT3 receptors (Fpeak/F0: 1.2760.06 in control vs 1.2560.06 with 2-Me-5-HT, n = 5) (the right trace in Fig. 2A). Also, in order to confirm the target cells, 2-Me-5-HT (10 mM) was applied in cell cluster preparations from W/Wv mice, but no [Ca2+]i oscillation was induced (n = 5). O-methyl-5-HT (O-Me-5-HT) is a known non-selective 5-HT receptor agonist, but lacking affinity for 5-HT3 receptors. Application of O-Me-5-HT (10 mM) caused no recovery of spontaneous [Ca2+]i activity in the presence of LY-278584 (10 mM)(n = 4, Fig. 2B). Essentially the same results as shown in Fig 2A–B were obtained in the presence of TTX (250 nM), indicating that there is no contribution of neural activity: Namely these drugs work through action potential-independent mecha- nisms. In contrast to the effects of 5-HT3 antagonists, application of 10 mM GR113808, a selective antagonist for 5-HT4, had little effect on spontaneous [Ca2+]i oscillations in ICC (Fpeak/F0: 1.2560.06 in control vs 1.2260.02, n = 5)(middle trace in Fig. 2C). Additional application of 10 mM LY-278584 again suppressed [Ca2+]i activity (right trace in Fig. 2C). These pharmacological examinations, together suggest that pacemaker [Ca2+]i activity in ICC is generated by endogenous 5-HT via 5- HT3 receptors. Ca2+ influx sensitive to SK&F96365 plays an essential role in generating ICC pacemaker [Ca2+]i activity, presumably co- ordinating with intracellular Ca2+ release channels [16,17]. In order to assess whether this Ca2+ influx pathway contributes to the pacemaker activity mediated via 5-HT3 receptors, the effect of 2- Me-5-HT was examined in the presence of SK&F96365. Application of 40 mM SK&F96365 suppressed pacemaker [Ca2+]i activity in ICC as observed previously [17], but subsequent PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e24928