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First published online 1 September 2005
doi: 10.1242/jcs.02540

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Sulphonylurea receptors differently modulate ICC pacemaker Ca²⁺ activity and smooth muscle contractility

¹ Department of Cell Physiology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
² Department of Physiological Medicine,Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
³ Department of Anatomy and Cell Biology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan
⁴ Department of Molecular and Cellular Pharmacology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
⁵ Department of Internal Medicine, Aichi-Gakuin University School of Dentistry, Nagoya 464-8651, Japan
⁶ Biochemical Engineering of Environment Center, Faculty of Chemical Science and Engineering, University of Petroleum, Beijing 102249, China
⁷ Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, UK

View larger version (34K): [in a new window]   Fig. 1. Spontaneous [Ca2+]i oscillation seen in a c-Kit-immunopositive region of a cell cluster preparation in the presence of cromakalim. (a,b) Fluo-3 Ca2+ images acquired from a cell cluster preparation at basal and peak times of an oscillation cycle, respectively. The Ca2+ image in b was normalized to a [(a)=F0, (b)=Fpeak/F0]. This cell cluster preparation was treated with 3 µM cromakalim. (c,d) A transmission image of the same cell cluster and an immunostaining with an anti-c-Kit antibody, ACK2, respectively. This immunostaining was done after the [Ca2+]i measurement. Scale bars: (below b for a and b, and below d for c and d) 50 µm.

View larger version (35K): [in a new window]   Fig. 2. Simultaneous estimation of spontaneous [Ca2+]i oscillations and mechanical activity. (A) Ca2+ images obtained from a cell cluster preparation with a high intensity area that could be used to monitor mechanical activity. (a) The initial fluorescent Ca2+ image. (b1,b2) Pseudocoloured Ca2+ images acquired at basal and peak times, respectively, of an initial oscillation cycle in normal solution, while (c1) and (c2) are at basal and peak times of an initial oscillation cycle in the presence of cromakalim (1 µM). Unlike, the Ca2+ image in Fig. 1b, it was not possible to normalize the Ca2+ images, because of contractile activity in control normal solution. Scale bar: (below a) 50 µm. (B) The time course of [Ca2+]i oscillations (upper trace) was measured in the boxed region of the cell cluster preparation shown in Aa; left traces are in normal solution and right traces are with cromakalim. The size and position of this region were chosen in order to minimize the interference from mechanical activity in control solution. The Fluo-3 fluorescence is expressed relative to that at the initial basal time (Ft/F0(t=ib)). The mechanical activity (lower trace) was simultaneously monitored by tracking the high-intensity area indicated by the arrow in Aa. (C) Three successive oscillation cycles in control solution are shown expanded in order to clearly show relationship between [Ca2+]i and mechanical activities.

View larger version (18K): [in a new window]   Fig. 3. Effects of various concentrations of cromakalim on the [Ca2+]i oscillation frequency. Changes in the frequency are plotted for individual cell cluster preparations. The graph in A shows the effects of 1 (filled circles) or 3 µM cromakalim (open circles) applied in normal extracellular medium. In B, 10 µM cromakalim (filled triangles) was used in the presence of 1 µM nifedipine.

View larger version (30K): [in a new window]   Fig. 4. Effects of cromakalim on the [Ca2+]i oscillation amplitude. The amplitude of [Ca2+]i oscillation is expressed relative to that before application of cromakalim. The experiments for 10 µM cromakalim (Crm) were carried out in the presence of nifedipine (1 µM). The number of experiments is shown in brackets. The effect of 10 µM cromakalim was significantly inhibited by a prior application of 1 µM glybenclamide (Glib) (P<0.05, indicated by an asterisk).

View larger version (17K): [in a new window]   Fig. 5. Interaction of cromakalin, glibenclamide and high-K+ treatments. In A, [Ca2+]i oscillations (Ft/F0(t=ib)) in a pacemaker region was measured in the presence of nifedipine (1 µM) and glibenclamide (1 µM) (a). Subsequently, 10 µM cromakalim was added (b: after 3 minutes). In B, after observing a control spontaneous [Ca2+]i oscillations in the presence of 1 µM nifedipine (a), [Ca2+]i oscillation traces (b) and (c) were obtained 3 and 5 minutes after application of cromakalim (10 µM), respectively. Subsequently, the extracellular K+ concentration was increased to 15.9 mM (from 5.9 mM) in the presence of cromakalim, and the trace (d) was obtained after 3 minutes.

View larger version (21K): [in a new window]   Fig. 6. Effects of diazoxide. After observing control [Ca2+]i oscillations in the presence of nifedipine (1 µM), diazoxide was cumulatively added. Trace (b) was recorded 3 minutes after application of 10 µM diazoxide, and (c) was 1 min after application of 30 µM diazoxide. Subsequently, the extracellular K+ concentration was increased to 15.9 mM. The trace (d) was obtained after 3 minutes.

View larger version (24K): [in a new window]   Fig. 7. RT-PCR examinations of effectors for K+ channel openers. RNA samples were obtained from isolated c-Kit-immunopositive cells (ICCs) or smooth muscle cells (SMCs). NTC represents `no template control'. RT-PCR was performed with six-pair primers for 45 (Kir6.1, Kir6.2, SUR1. SUR2, and c-kit) or 40 (GAPDH) cycles. Amplified products were separated on 2.5% agarose gels and analyzed by ethidium bromide staining. The numbers in the right of each gel indicate the size marker (bp). GAPDH was amplified as an index of proper amplification, and c-kit was examined in order to confirm the c-Kit-immunoreactivity under the fluorescent microscope.

View larger version (149K): [in a new window]   Fig. 8. Immunohistochemistry for c-Kit and SUR. Smooth muscle layer (including the myenteric plexus) of ileum was double-labelled with anti-c-Kit antibody (A: ACK2 with Alexa Fluor 488, red) and anti-SUR1 antibody (B: C16 with Alexa Fluor 594, green); C shows a merged image. Note that network-forming cells were stained yellow, indicating that ICCs in the ileum express SUR1. Scale bar: 20 µm. (D) An enlarged view of the boxed region in C. A single cell with both anti-c-Kit and anti-SUR1 immunoreactivity is indicated by a dotted lines.

View larger version (38K): [in a new window]   Fig. 9. Schematic diagram showing possible modulation mechanisms on smooth muscle contractility and ICC pacemaker [Ca2+]i oscillations via SUR. Both smooth muscle and ICCs express KATP channels, but different SUR isoforms are associated: SUR1 in ICCs and SUR2B in smooth muscle. Under normal conditions, ryanodine receptors (RyR) and InsP3 receptors (InsP3R) in the endoplasmic reticulum (ER) co-ordinately produce pacemaker [Ca2+]i oscillations in ICCs, thereby pacemaker potentials are generated by periodic activation of plasmalemmal Ca2+-activated Cl– channels. Pacemaker potentials are conducted toward smooth muscle cells via gap junction channels. Consequently, voltage-dependent Ca2+ channels (VDCC) (mainly DHP-sensitive, L-type) are periodically activated to cause spontaneous phasic contractions in the gut. Applications of potent SUR2B activators, such as cromakalim, activate KATP channels in smooth muscle. Resultant hyperpolarization in the smooth muscle cell membrane prevents the periodic activation of VDCC, and suppresses spontaneous contractions, whereas pacemaker [Ca2+]i oscillations remain in ICCs. When KATP channel openers that act on SUR1, such as diazoxide, are applied, ICC pacemaker [Ca2+]i oscillations are suppressed in a voltage-independent manner. Unknown intracellular signals may link between SUR1 and activity of intracellular Ca2+ release channels. Mitochondrial signals might be also involved. The predominant expression of SUR1 in ICCs can account for the fact that spontaneous rhythmicity (electrical activity) in the gut smooth muscle tissues is highly dependent upon energy metabolism (e.g. Tomita, 1981; Nakayama et al., 1997).

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