Properties of pacemaker potentials recorded from myenteric interstitial cells of Cajal distributed in the mouse small intestine

doi:10.1113/jphysiol.2003.051334

Properties of pacemaker potentials recorded from myenteric interstitial cells of Cajal distributed in the mouse small intestine

Yoshihiko Kito and Hikaru Suzuki

Department of Physiology, Nagoya City University Medical School, Mizuho-ku, Nagoya 467-8601, Japan

ABSTRACT

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Abstract
Introduction
Methods
Results
Discussion
References

Recording of electrical responses from isolated small intestine of mice using conventional microelectrodes revealed two types of potential, a pacemaker potential and a slow wave, both with rapid rising primary components and following plateau components. The rate of rise and peak amplitude were greater for pacemaker potentials than for slow waves, and the plateau component was smaller in slow waves than in pacemaker potentials. Both potentials oscillated at a similar frequency (20-30 min^-1). Unitary potentials often discharged during the interval between pacemaker potentials. Infusion of Lucifer Yellow allowed visualization of the recorded cells; pacemaker potentials were recorded from myenteric interstitial cells of Cajal (ICC-MY) while slow waves were recorded from circular smooth muscle cells. Pacemaker potentials were characterized as follows: the primary component was inhibited by Ni²⁺, Ca²⁺-free solution or depolarization with high-K⁺ solution, the plateau component was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), an inhibitor of Ca²⁺-activated Cl^- channels, low [Cl^-]_o solution or Ca²⁺-free solution, and the generation of potentials was abolished by co-application of Ni²⁺and DIDS or by chelating intracellular Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM). These results indicate that in the mouse small intestine ICC-MY generate pacemaker potentials with two components in situ; the primary and plateau components may be generated by activation of voltage-dependent Ca²⁺-permeable channels and Ca²⁺-activated Cl^- channels, respectively. Slow waves are generated in circular smooth muscles via electrotonic spread of pacemaker potentials. These properties of intestinal pacemaker potentials are considered essentially similar to those of gastric pacemaker potentials.

(Received 15 July 2003; accepted after revision 13 October 2003; first published online 17 October 2003)
Corresponding author H. Suzuki: Department of Physiology, Nagoya City University Medical School Mizuho-ku, Nagoya 467-8601, Japan. Email: hisuzuki{at}med.nagoya-cu.ac.jp

INTRODUCTION

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Abstract
Introduction
Methods
Results
Discussion
References

Smooth muscles forming the gastrointestinal tract are spontaneously active with rhythmic generation of slow waves or action potentials or both (Tomita, 1981). These activities may originate in interstitial cells of Cajal (ICC) distributed in the myenteric region (ICC-MY), rather than in smooth muscle cells (Thuneberg, 1982; Sanders, 1996), since the rhythmic activity is strongly attenuated in the small intestine of animal models lacking ICC-MY (Maeda et al. 1992; Ward et al. 1994; Huizinga et al. 1995). This could explain the causal relationship of the reduced number or loss of ICC-MY in patients suffering from a variety of disorders of gastrointestinal motility (Huizinga et al. 1997; Sanders et al. 1999; Vanderwinden & Rumessen, 1999). ICC express Kit receptors and form gap junctional connections with surrounding ICC and smooth muscle cells (Torihashi et al. 1995; Komuro et al. 1996; Sanders et al. 1999). An ongoing discharge of rhythmic electrical activity generated in ICC-MY is thought to pace smooth muscle activity by passive propagation, which then activates a variety of ionic channels to excite smooth muscles (Dickens et al. 1999; Sanders et al. 1999; Dickens et al. 2000; Hirst & Edwards, 2001; Koh et al. 2002). In addition, recent studies indicate that an expression of functional receptors for inositol 1,4,5-trisphosphate (IP₃) (Suzuki et al. 2000) and coupling of Ca²⁺ release from IP₃-sensitive Ca²⁺ stores and Ca²⁺ uptake into mitochondria may be key factors for the rhythmic generation of pacemaker activity (Ward et al. 2000; Fukuta et al. 2002).

Cultured ICC-MY isolated from the mouse small intestine produce a rhythmic generation of inward currents by activation of Cl^- channels (Tokutomi et al. 1995; Huizinga et al. 2002) or non-selective cation channels (Koh et al. 1998; Thomsen et al. 1998; Nakayama & Torihashi, 2002). However, it remains unclear whether this is indeed the case for ICC-MY in situ, since the activity of functional proteins expressed at the plasma membrane is often altered during the culture of cells (Snetkov et al. 1996; Sui et al. 2001; Ihara et al. 2002). In intact tissues of the mouse small intestine, the pacemaker activity of ICC-MY could be detected indirectly from circular smooth muscle cells as slow waves, and all estimates of the properties of pacemaker potentials have been made from slow waves (Ward et al. 1994; Huizinga et al. 1995; Malysz et al. 2001). Therefore, analysis of the ionic mechanisms underlying pacemaker potentials may require direct recording of potentials from ICC-MY, as in the case of the stomach of guinea-pig, in which ICC-MY generate large amplitude pacemaker potentials in situ (Dickens et al. 1999). Gastric pacemaker potentials consist of two components, a rapidly rising primary component and a subsequent long-lasting plateau component (Dickens et al. 1999, 2000; Hirst & Edwards, 2001; Kito et al. 2002a; Kito & Suzuki, 2003a,b). Similar forms of pacemaker potentials have also been recorded from the mouse gastric antrum (Hirst et al. 2002b). The primary and plateau components of pacemaker potentials may be produced by an opening of voltage-dependent Ca²⁺-permeable channels and Ca²⁺-activated Cl^- channels, respectively, and the primary component may be triggered by membrane depolarization due to summation of unitary potentials (Hirst & Edwards, 2001; Kito et al. 2002c; Kito & Suzuki, 2003a).

Intracellular recordings of electrical activity from the mouse small intestine revealed two types of periodically generated potential: slow waves recorded from circular smooth muscle cells with amplitudes over 20 mV having no superimposed Ca²⁺ spikes on the top, and bursts of Ca²⁺ spikes generated during small (< 20 mV) depolarizations recorded from longitudinal smooth muscle cells (Ward et al. 1994; Huizinga et al. 1995; Sanders, 1996; Takano & Suzuki, 2001). Here, attempts were made to characterize the electrophysiological properties of pacemaker potentials recorded from the mouse small intestine. Experiments were carried out by recording the potentials directly from ICC-MY using intracellular recording techniques, and the recorded cells were visualized by injecting a dye, Lucifer Yellow. The results indicated that in the mouse small intestine ICC-MY generate pacemaker potentials with two components, a primary component forming a rapid rising phase and a following plateau component. The primary component may be generated by activation of voltage-dependent Ca²⁺-permeable channels and the plateau component by opening of Ca²⁺-activated Cl^- channels. Slow waves, generated in circular smooth muscle cells, were considered to result from passive electrotonic propagation of pacemaker potentials from ICC-MY. The similarities and differences in the properties of pacemaker potentials between mouse small intestine and guinea-pig gastric antrum are also discussed.

METHODS

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Abstract
Introduction
Methods
Results
Discussion
References

BALB/c mice of either sex, aged 4-6 weeks, were anaesthetized with fluoromethyl 2, 2, 2-trifluoro-1-(trifluoromethyl) ethyl ether (sevoflurane, Maruishi Pharm., Osaka, Japan), and killed by cervical dislocation and exsanguination. All animals were treated ethically according to the Guidelines for the Care and Use of Laboratory Animals of Nagoya City University Medical School, acredited by The Physiological Society of Japan. Segments of terminal ileum were removed from animals and opened along the mesenteric border, in Krebs solution (see below). The mucosal layers, the serosal layers and a part of the longitudinal layers were carefully peeled away under a dissecting microscope. A tissue segment (about 0.5 mm wide and 0.5 mm long) was pinned out on a silicone rubber plate with the serosal side uppermost, and the plate was fixed at the bottom of an organ bath (8 mm wide, 8 mm deep, 20 mm long). The tissue was superfused with warmed (35 °C) and oxygenated Krebs solution, at a constant flow rate of about 2 ml min^-1. Experiments were carried out in the presence of 3 µM nifedipine throughout, and this minimized the movement of muscles.

Conventional microelectrode techniques were used to record intracellular electrical responses from smooth muscle tissues, and the glass capillary microelectrodes (outer diameter 1.2 mm, inner diameter 0.6 mm; Hilgenberg, Germany) filled with 3 M KCl had tip resistances ranging between 50 and 80 M $Omega$ . Electrical responses recorded via a high input impedance amplifier (Axoclamp-2B, Axon Instruments, USA) were displayed on a cathode ray oscilloscope (SS-7602, Iwatsu, Osaka, Japan) and also stored on a personal computer for later analysis.

To identify the morphological properties of cells in the small intestine, cells were impaled with microelectrodes filled with 1 M LiCl and 5 % w/v Lucifer Yellow (Sigma, USA). The impaled cell was labelled with Lucifer Yellow by passing hyperpolarizing current pulses (duration 100 ms, intensity 1 nA, frequency 3 Hz for 2-30 min) supplied from an electric stimulator (SEN-3301, Nihon Kohden, Tokyo, Japan). Preparations filled with Lucifer Yellow were fixed overnight at 4^oC with fresh 4 % w/v paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). The next day, the preparations were washed several times with PBS, mounted in DAKO fluorescent mounting medium (DAKO Corporation, CA, USA), covered with a coverslip and viewed with a confocal microscope (LSM5 PASCAL, Carl Zeiss, Germany). The confocal microscope with a krypton-argon laser allowed the visualization of Lucifer Yellow (458 nm excitation filter and 475 nm emission long pass filter).

The ionic composition of the Krebs solution was as follows (mM): Na⁺, 137.4; K⁺, 5.9; Ca²⁺, 2.5; Mg²⁺, 1.2; HCO₃^-, 15.5; H₂PO₄^-, 1.2; Cl^- , 134; glucose, 11.5. Solutions containing high-potassium ion concentrations (high K⁺ solutions) were prepared by replacing NaCl with KCl. Low Cl^- solution ([Cl^-]_o = 13.3 mM) was prepared by equimolar replacement of NaCl with sodium isethionate. The solutions were aerated with O₂ containing 5 % CO₂, and the pH of the solutions was maintained at 7.2-7.3.

Drugs used were atropine sulphate, 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS), forskolin, 3-morpholino-sydnonimine (SIN1), nifedipine, N-nitro-L-arginine (L-NA), tetrodotoxin (all from Sigma, USA), and 1,2-bis(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) (from Dojindo, Osaka, Japan). Forskolin, nifedipine and SIN1 were dissolved in dimethyl sulphoxide (DMSO) to make stock solutions, and were added to Krebs solution to make the desired concentrations, just prior to use. Other drugs tested were dissolved in distilled water. The final concentration of the solvent in Krebs solution did not exceed 1/1000. Addition of these chemicals to Krebs solution did not alter the pH of the solution.

Experimental values are expressed as the mean value ± standard deviation (S.D.). Statistical significance was tested using Student's t test, and probabilities of less than 5 % (P < 0.05) were considered significant.

RESULTS

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Abstract
Introduction
Methods
Results
Discussion
References

Spontaneous electrical activities recorded from intact tissue preparation of mouse small intestine

In preliminary experiments, access to ICC-MY with microelectrodes was unsuccessful in preparations with an attached intact longitudinal muscle layer. Therefore attempts were made to remove not only the serosa but also a part of the longitudinal layers carefully from the tissue under a dissecting microscope, and this facilitated the impalement of ICC-MY. In such preparations, most of the impaled cells generated slow waves (Fig. 1A). The membrane potential of cells generating slow waves at its most negative value (equal to the resting membrane potential) ranged between -61.7 and -77.7 mV (mean -69.9 ± 3.9 mV, n = 34; each n value represents the number of animals used). Slow waves had two components: an initial transient depolarization forming a primary component and a following slow depolarization which formed a plateau (Fig. 1Da). The primary component of slow waves had amplitudes ranging between 26.5 and 45.1 mV (mean 34.1 ± 4.6 mV; n = 34) and the plateau component had peak amplitudes ranging between 18.0 and 40.2 mV (mean 31.1 ± 4.4 mV; n = 34). Slow waves had a mean half-width (the duration of potential measured at the half-amplitude of the peak) of 1.07 ± 0.20 s (n = 34) and frequency of 26.1 ± 2.8 min^-1 (n = 34). The mean value of the maximum rate of rise (dV/dt_max) of the primary component was 0.54 ± 0.10 V s^-1 (n = 34), while the mean dV/dt_max of the plateau component was 0.09 ± 0.03 V s^-1 (n = 34).

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Figure 1. Slow waves and pacemaker potentials recorded from mouse small intestine
A, a train of slow waves. B, a train of pacemaker potentials. C, a noisy pattern of pacemaker potentials. D, superimposed slow wave (a) and pacemaker potential (b) recorded at high speed (recorded from the same preparation). The resting membrane potentials were: A -77 mV, B -76 mV, C -66 mV, D -76 mV. A-D were recorded from different tissues.

A different type of rhythmic potential profile was also recorded, but in less than 1 % of all the impalements. These potentials had a rapid rising phase (primary component) and following plateau component with monotonically declining amplitude (Fig. 1B). The initial primary component had an amplitude ranging between 47.4 and 68.1 mV (mean 56.5 ± 5.3 mV; n = 39), the value being significantly larger than that of slow waves (P < 0.01). The dV/dt_max of the primary component had a mean value of 1.51 ± 0.30 V s^-1 (n = 39), the value being again significantly larger than that of slow waves (P < 0.01). These potentials had a mean half-width of 1.00 ± 0.16 s (n = 39) and frequency of 26.1 ± 2.4 min^-1 (n = 39), and each of these values was not significantly different from those of slow waves (P > 0.05). The resting membrane potential of these cells ranged between -62.5 and -78.9 mV (mean -69.6 ± 4.5 mV, n = 39), the values being not significantly different from those of cells with slow waves (P > 0.05). Thus as the properties of these potentials were similar to those of 'driving potentials' recorded from ICC-MY in gastric antrum (Dickens et al. 1999), the term 'pacemaker potential' was applied to these potentials. On some occasions, discharges of membrane noise with irregular amplitude appeared during the falling phase (repolarizing phase) of pacemaker potentials (Fig. 1C). Unitary potentials, or a similar type of potential, found in gastric muscles (Suzuki & Hirst, 1999) were also observed during the interval between pacemaker potentials (Fig. 3A), but their appearance was infrequent. A superimposed comparison of a pacemaker potential and a slow wave shows the characteristic differences between these two potentials (Fig. 1Da and b).

Morphological properties of cells generating slow waves and pacemaker potentials

Attempts were made to visualize spontaneously active cells by injecting the dye Lucifer Yellow. After impalement, hyperpolarizing currents (intensity, 1 nA; duration, 100 ms; 3 Hz frequency for 2-30 min) were applied to the electrode to inject Lucifer Yellow. The results indicated that injection of Lucifer Yellow for a short period of time (< 5 min) into a cell from which slow waves had been recorded labelled a spindle-shaped cell that had the morphological characteristics of smooth muscle cells (Fig. 2B) (n = 5). When Lucifer Yellow was injected for more than 20 min to cells with slow waves (Fig. 2Aa) (n = 10), the dye diffused to surrounding cells distributed about 500 µm away in the longitudinal direction and about 50 µm away in the transverse direction (Fig. 2Ab and c). All cells with slow waves distributed in circular muscle bundles had lengths between 200 and 300 µm and maximal widths of about 10 µm. These values were comparable to those of circular smooth muscle cells of the mouse small intestine (Komuro, 1999), suggesting that slow waves were generated in circular smooth muscle cells.

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Figure 2. Morphological properties of slow wave generating cells after infusion of Lucifer Yellow
Aa, slow waves recorded from a cell shown in Ab. The resting membrane potential was -65 mV. The preparation was viewed with a confocal microscope (Ab). Ac is an enlarged view of part of Ab. B is a confocal image of another preparation loaded with Lucifer Yellow. The calibration bars on Ab, Ac and B represent 200 µm, 50 µm and 100 µm, respectively.

In a different series of experiments, cells with pacemaker potentials were visualized by infusing Lucifer Yellow. A brief injection of Lucifer Yellow (< 2 min) allowed visualization of only a few cells (n = 3; data not shown). On the other hand, injection of Lucifer Yellow for a long period of time (> 20 min) allowed the dye to spread to cells over 500 µm away from the injected cell in all directions on a single plane (Fig. 3Ab and c) (n = 12). The nuclei of cells had the tendency to be stained more brightly than the cytoplasm. These cells were distributed in the myenteric layer, between the circular and longitudinal muscle layers, and each cell possessed an oval or triangular cell body with several (two to five) fine processes (Fig. 3B). The cell bodies had variable diameters, ranging between small types (< 10 µm) and large types (> 20 µm). The length of the processes was also variable, with short (< 10 µm) and long (> 100 µm) processes. These processes made contact with neighbouring cells to form a network. These morphological characteristics were quite similar to those of ICC-MY distributed in small intestine but completely distinct from smooth muscle cells (Thuneberg, 1982; Torihashi et al. 1995; Komuro et al. 1996; Sanders, 1996; Dickens et al. 1999; Belzer et al. 2002). These results suggest that pacemaker potentials were recorded from ICC-MY of the mouse small intestine.

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Figure 3. Morphological properties of pacemaker potential generating cells after infusion of Lucifer Yellow
Aa, pacemaker potentials recorded from a cell shown in Ab. The resting membrane potential was -70 mV. The fluorescence of the injected Lucifer Yellow was viewed with a confocal microscope (Ab). Ac is an enlarged view of Ab, in which the impaled cell is indicated by the arrow. B is a confocal image of another preparation loaded with Lucifer Yellow. The calibration bars on Ab, Ac and B represent 100 µm, 20 µm and 20 µm, respectively.

Effects of NiCl₂, DIDS and low [Cl^-]_o solution on pacemaker potentials

The effects of NiCl₂ on pacemaker potentials recorded from ICC-MY of mouse small intestine were investigated, since slow waves recorded from circular muscles of canine colon are abolished by 40 µM NiCl₂ in the presence of nifedipine (Ward & Sanders 1992b) and 100 µM NiCl₂ decreased the dV/dt_max of pacemaker potentials recorded from the guinea-pig gastric antrum (Tomita et al. 1998). NiCl₂ (10-60 µM) decreased the frequency and the dV/dt_max of pacemaker potentials in a concentration-dependent manner (Fig. 4A-D; Table 1). NiCl₂ (10-60 µM) had no effect on the resting membrane potentials or on the amplitudes of pacemaker potentials (Table 1). As shown in Fig. 4E, NiCl₂ (60 µM) selectively abolished the initial rapid depolarization without affecting other electrophysiological characteristics of pacemaker potentials (Table 1). In three preparations, unitary potentials were generated during the intervals between pacemaker potentials after application of 60 µM NiCl₂.

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Figure 4. Effects of NiCl₂ on pacemaker potentials recorded from mouse small intestine
Pacemaker potentials were recorded before (A) and during application of NiCl₂ (B, 10 µM; C, 30 µM; D, 60 µM). E, high speed traces of pacemaker potentials recorded in the absence (a) and presence of 60 µM NiCl₂ (b). All traces were recorded from the same cell with a resting membrane potential of -75 mV.

It has been reported that in the guinea-pig gastric antrum, the plateau component of pacemaker potentials is inhibited by DIDS, a blocker of Ca²⁺-activated Cl^- channels, or by low [Cl^-]_o solution (Kito et al. 2002a; Kito & Suzuki, 2003a). A possible involvement of chloride conductance in the generation of intestinal pacemaker potentials was tested by observing the effects of DIDS and low [Cl^-]_o solution on pacemaker potentials. DIDS (2 mM) hyperpolarized the membrane (3.9 ± 1.4 mV, n = 7) and reduced the half-widths of pacemaker potentials (control, 1.13 ± 0.21 s; in DIDS, 0.45 ± 0.08 s; n = 7; P < 0.01), with no significant alteration to their amplitudes (control, 58.0 ± 5.9 mV; in DIDS, 59.9 ± 5.0 mV; n = 7; P > 0.05), frequency (control, 25.1 ± 2.9 min^-1; in DIDS, 27.0 ± 1.4 min^-1; n = 7; P > 0.05) or dV/dt_max (control, 1.32 ± 0.22 V s^-1; in DIDS, 1.25 ± 0.24 V s^-1; n = 7; P > 0.05) (Fig. 5A and B). In the presence of low [Cl^-]_o solution, the frequency (control, 25.2 ± 2.8 min^-1; in low [Cl^-]_o solution, 18.9 ± 3.5 min^-1; n = 8; P < 0.01) and half-width (control, 1.04 ± 0.19 s; in low [Cl^-]_o solution, 0.53 ± 0.07 s; n = 8; P < 0.01) of pacemaker potentials were decreased, with no significant alteration to their amplitude (control, 56.0 ± 5.5 mV; in low [Cl^-]_o solution, 58.7 ± 3.6 mV; n = 8; P > 0.05) or dV/dt_max (control, 1.32 ± 0.26 V s^-1; in low [Cl^-]_o solution, 1.22 ± 0.36 V s^-1; n = 8; P > 0.05) (Fig. 5C and D). These results indicated that DIDS and low [Cl^-]_o solution decreased the half-width of pacemaker potentials without altering the initial primary component.

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Figure 5. Effects of DIDS and low [Cl^-]_o solution on pacemaker potentials recorded from mouse small intestine
Pacemaker potentials were recorded before (Aa) and during application of 2 mM DIDS (Ab). B, high speed traces of pacemaker potentials recorded in the absence (a) and presence of 2 mM DIDS (b). Pacemaker potentials were recorded before (Ca) and during application of low [Cl^-]_o solution (Cb). D, high speed traces of pacemaker potentials recorded in the absence (a) and presence of low [Cl^-]_o solution (b). The resting membrane potentials were: A -65 mV, C -70 mV. A and C were recorded from different tissues.

To characterize further the primary and plateau components of pacemaker potentials, the effects of the combined application of NiCl₂, DIDS and low [Cl^-]_o solution on pacemaker potentials were studied. In the presence of 60 µM NiCl₂, additional application of 2 mM DIDS abolished pacemaker potentials with no alteration in the resting membrane potential (in NiCl₂, -70.9 ± 5.5 mV; in NiCl₂ and DIDS, -71.1 ± 5.5 mV; n = 5; P > 0.05) (Fig. 6A). Similarly, in the presence of 2 mM DIDS, additional application of 60 µM NiCl₂ again abolished pacemaker potentials without altering the resting membrane potential (in DIDS, -68.9 ± 5.0 mV; in NiCl₂ and DIDS, -68.7 ± 5.2 mV; n = 5; P > 0.05) (Fig. 6B). In the presence of low [Cl^-]_o solution, 60 µM NiCl₂ reduced the frequency of pacemaker potentials (in low [Cl^-]_o solution, 18.7 ± 1.9 min^-1; in low [Cl^-]_o solution and NiCl₂, 3.3 ± 1.6 min^-1; n = 4; P < 0.01), without altering the resting membrane potential (in low [Cl^-]_o solution, -66.7 ± 5.6 mV; in low [Cl^-]_o solution and NiCl₂, -66.6 ± 6.0 mV; n = 4; P > 0.05) (Fig. 6C).

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Figure 6. Effects of combined application of NiCl₂, DIDS and low [Cl^-]_o solution on pacemaker potentials recorded from mouse small intestine
A, DIDS (2 mM) was applied to preparation (indicated by the horizontal bar) in the presence of 60 µM NiCl₂. B, NiCl₂ (60 µM) was applied to preparation (indicated by the horizontal bar) in the presence of 2 mM DIDS. C, NiCl₂ (60 µM) was applied to preparation (indicated by the horizontal bar) in the presence of low [Cl^-]_o solution. The resting membrane potentials were: A -69 mV, B -72 mV, C -69 mV. All traces were recorded from different tissues.

Taken together, these results suggest that a pacemaker potential recorded from ICC-MY has two components: a rapidly rising Ni²⁺-sensitive primary component and a following plateau component that is dependent upon Cl^- conductance. The results also suggest that the primary and plateau components are generated by different mechanisms, since each of them could be generated independently.

Properties of slow waves recorded from circular smooth muscle cells

The effects of NiCl₂, DIDS and low [Cl^-]_o solution on slow waves were also studied, to characterize properties of each component. NiCl₂ (60 µM) decreased the frequency (control, 24.2 ± 2.7 min^-1; in NiCl₂, 16.6 ± 1.4 min^-1; n = 7; P < 0.01) and dV/dt_max (control, 0.60 ± 0.08 V s^-1; in NiCl₂, 0.11 ± 0.03 V s^-1; n = 7; P < 0.01) of slow waves, with no alteration in the resting membrane potential (control, -68.5 ± 3.7 mV; in NiCl₂, -66.4 ± 4.4 mV; n = 7; P > 0.05) or the amplitude of the plateau component of slow waves (control, 31.2 ± 4.1 mV; in NiCl₂, 32.1 ± 3.4 mV; n = 7; P > 0.05) (Fig. 7A). In the presence of 60 µM NiCl₂, the duration of slow waves was significantly increased (control, 0.94 ± 0.15 s; in NiCl₂, 1.32 ± 0.19 s; n = 7; P < 0.01). On the other hand, DIDS (2 mM) reduced the amplitude of both primary (control, 30.9 ± 3.2 mV; in DIDS, 23.5 ± 5.2 mV; n = 6; P < 0.05) and plateau (control, 26.6 ± 2.0 mV; in DIDS, 16.1 ± 3.0 mV; n = 6; P < 0.01) components, the duration (control, 1.05 ± 0.13 s; in DIDS, 0.56 ± 0.07 s; n = 6; P < 0.01) and the dV/dt_max (control, 0.56 ± 0.07 V s^-1; in DIDS, 0.38 ± 0.08 V s^-1; n = 6; P < 0.01) of slow waves, with no significant alteration in the frequency (control, 24.3 ± 3.5 min^-1; in DIDS, 26.3 ± 5.1 min^-1; n = 6; P > 0.05) (Fig. 7B). DIDS (2 mM) hyperpolarized the membrane by 3.8 ± 1.7 mV (n = 6). Application of low [Cl^-]_o solution significantly reduced the amplitude of the plateau component (control, 28.0 ± 3.6 mV; in low [Cl^-]_o solution, 18.2 ± 4.1 mV; n = 7; P < 0.01), the frequency (control, 26.6 ± 2.3 min^-1; in low [Cl^-]_o solution, 20.6 ± 2.8 min^-1; n = 7; P < 0.01), the duration (control, 1.03 ± 0.18 s; in low [Cl^-]_o solution, 0.50 ± 0.15 s; n = 7; P < 0.01) and the dV/dt_max (control, 0.51 ± 0.09 V s^-1; in low [Cl^-]_o solution, 0.36 ± 0.14 V s^-1; n = 7; P < 0.01) of slow waves, with no alteration in the amplitude of the primary component (control, 32.4 ± 3.5 mV; in low [Cl^-]_o solution, 31.8 ± 4.3 mV; n = 7; P > 0.05) (Fig. 7C). These results indicate that NiCl₂ is a selective inhibitor of the primary component of slow waves, as in the case of pacemaker potentials (Fig. 6). DIDS and low [Cl^-]_o solution both inhibited the plateau component, and DIDS also inhibited the primary component.

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Figure 7. Properties of two components of slow waves recorded from circular smooth muscle cells of mouse small intestine and the relationship between transient repolarization and dV/dt_max of slow waves
A, high speed traces of slow waves recorded in the absence (a) and presence of 60 µM NiCl₂ (b). B, high speed traces of slow waves recorded in the absence (a) and presence of 2 mM DIDS (b). C, high speed traces of slow waves recorded in the absence (a) and presence of low [Cl^-]_o solution (b). D, large (a) and small (b) transient repolarizations after depolarization of primary component of slow waves. E, the relationship between dV/dt_max of slow waves (abscissa) and the amplitude of repolarization (ordinate). The regression line is given by Y = 0.08 + 28.5X (Y, amplitude of repolarization; X, dV/dt_max; r = 0.75, n = 128 from 8 tissues, P < 0.0001). The resting membrane potentials were: A -71 mV, B -70 mV, C -65 mV, D -72 mV. All traces were recorded from different tissues.

The amplitude of the repolarization phase that occurs between the initial primary and following plateau components of slow waves varied between individual events, even within a record taken from a single impalement (Fig. 7D). The possibility of a causal relationship between the amplitude of repolarization and the primary component of slow waves was investigated. When the amplitude of repolarization was plotted as a function of the dV/dt_max of the primary component of slow waves, these two factors were found to be positively related (Fig. 7E). The regression line calculated by using the least-squares method was given by Y = 0.08 + 28.5X (Y, amplitude of repolarization; X, dV/dt_max), with a correlation coefficient (r) of 0.75 (n = 128). The results indicate that the amplitude of repolarization is a function of the dV/dt_max of the primary component of slow waves.

Effects of nominally Ca²⁺- free solution and BAPTA-AM on pacemaker potentials

Experiments were carried out to observe the effects of nominally Ca²⁺-free solution on pacemaker potentials in the mouse small intestine. In eight of eleven preparations, application of nominally Ca²⁺-free solution hyper-polarized the membrane by 3.6 ± 1.1 mV transiently (2-4 min). In the presence of nominally Ca²⁺-free solution for over 10 min, reductions were observed in all parameters of pacemaker potentials: amplitude (control, 57.3 ± 7.4 mV; in nominally Ca²⁺-free solution, 44.9 ± 16.0 mV; n = 11; P < 0.05), frequency (control, 26.6 ± 3.3 min^-1; in nominally Ca²⁺-free solution, 14.0 ± 5.2 min^-1; n = 11; P < 0.01), duration (control, 0.96 ± 0.20 s; in nominally Ca²⁺-free solution, 0.60 ± 0.14 s; n = 11; P < 0.01) and dV/dt_max (control, 1.61 ± 0.44 V s^-1; in nominally Ca²⁺-free solution, 0.39 ± 0.18 V s^-1; n = 11; P < 0.01) (Fig. 8B). Application of nominally Ca²⁺-free solution depolarized the membrane by 6.9 ± 2.0 mV in five of eleven preparations (Fig. 8Ab). In ten of eleven preparations, unitary potentials were generated during the intervals between pacemaker potentials in the presence of nominally Ca²⁺-free solution (Fig. 8Ab). In the presence of 2 mM DIDS, exposure of tissue to nominally Ca²⁺-free solution abolished pacemaker potentials within 4 min, with subsequent depolarization of the membrane by 4.9 ± 1.2 mV (n = 5) (Fig. 8C). These results suggest that the primary component of pacemaker potentials is generated by activation of Ca²⁺-permeable channels. It can again be seen that the primary and plateau components are generated independently, since as shown in Fig. 8Ab, the plateau component could be generated in the absence of the primary component.

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Figure 8. Effects of [Ca²⁺]_o-free solution on pacemaker potentials recorded from mouse small intestine
A, pacemaker potentials were recorded before (a) and during application of [Ca²⁺]_o-free solution (b). B, high speed traces of pacemaker potentials recorded in the absence (a) and presence of [Ca²⁺]_o-free solution for 22 min (b). C, [Ca²⁺]_o-free solution was applied to the preparation (indicated by the horizontal bar) in the presence of 2 mM DIDS. The resting membrane potentials were: A -70 mV, C -72 mV. A and C were recorded from different tissues.

The effects of BAPTA-AM on pacemaker potentials were observed to test the importance of intracellular Ca²⁺ handling mechanisms on the generation of pacemaker potentials in ICC-MY of mouse small intestine. Exposure of tissues to a solution containing 50 µM BAPTA-AM caused an initial inhibition of the primary component (Fig. 9Bb), and subsequent inhibition of plateau components to about one-tenth of control amplitude (Fig. 9Bc), before the complete disappearance of pacemaker potentials within 10-50 min (Fig. 9Bd). In the presence of BAPTA-AM, unitary potentials were generated in four out of eleven preparations (Fig. 9A and Bb). In six out of eleven preparations, BAPTA-AM depolarized the membrane by 6.1 ± 2.9 mV. These results suggest that availability of intracellular Ca²⁺ is required for generation of pacemaker potentials in ICC-MY of the mouse small intestine.

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Figure 9. Effects of BAPTA-AM on pacemaker potentials recorded from mouse small intestine
A, BAPTA-AM (50 µM) was applied (indicated by the horizontal bar). B, responses were recorded before (a) and during the application of 50 µM BAPTA-AM at 6 min (b), 12 min (c) and 37 min (d). The resting membrane potentials were: A -64 mV, B -69 mV. A and B were recorded from different tissues.

Effects of high K⁺ solution on pacemaker potentials recorded from ICC-MY

The effects of high K⁺ solution on pacemaker potentials generated in ICC-MY of the mouse small intestine were used to investigate the voltage dependency of pacemaker potentials. Application of high K⁺ solution depolarized the membrane and decreased the amplitude, frequency and dV/dt_max of pacemaker potentials in a concentration-dependent manner (Fig. 10A-D, Table 2). Similar results were obtained in the presence of tetrodotoxin (1 µM; n = 3), atropine (1 µM; n = 5) or L-NA (50 µM; n = 5) (data not shown). Interestingly, the duration of pacemaker potentials was decreased with 10.6 mM [K⁺]_o solution, and increased again with 20.0 mM [K⁺]_o solution (Table 2). The primary component of pacemaker potentials was inhibited by membrane depolarization with high K⁺ solution (Table 2). Furthermore, in the presence of 2 mM DIDS, depolarization of the membrane by 20.4 ± 1.3 mV (n = 5) with 15.3 mM [K⁺]_o solution abolished pacemaker potentials (data not shown), supporting the idea that the primary component of pacemaker potentials is a highly potential-dependent phenomenon. Unitary potentials, which were rarely detected in normal Krebs solution, appeared when the tissue was exposed to 15.3 mM [K⁺]_o solution or 20.0 mM [K⁺]_o solution (Fig. 10C and D). The shape of pacemaker potentials generated in the presence of 20.0 mM [K⁺]_o solution (Fig. 10D) resembled that of slow potentials recorded from circular muscle bundles of the guinea-pig gastric antrum with no attached ICC-MY (Suzuki & Hirst, 1999). These results indicate that the primary and plateau components of pacemaker potentials have different voltage dependencies, and the primary component disappears at smaller depolarizations. Furthermore, the results again confirmed that the plateau component could be generated in the absence of the primary component, as in the case of experiments with NiCl₂ (Fig. 4) or nominally Ca²⁺-free solution (Fig. 8).

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Figure 10. Effects of high K⁺ solution on pacemaker potentials recorded from mouse small intestine
Pacemaker potentials were recorded before (A) and during application of high K⁺ solution (B, 10.6 mM [K⁺]_o; C, 15.3 mM [K⁺]_o; D, 20.0 mM [K⁺]_o). All responses were recorded from the same cell with a resting membrane potential of -67 mV.

Effects of forskolin and SIN1 on pacemaker potentials and slow waves

The effects of forskolin and SIN1 on pacemaker potentials and slow waves were further investigated, since these cyclic nucleotide-related compounds inhibit pacemaker currents in cultured ICC-MY of the mouse small intestine (Koh et al. 2000). In ICC-MY, forskolin (5 µM) hyperpolarized the membrane and reduced the frequency and duration of pacemaker potentials, with no alteration in the amplitude and dV/dt_max, while in circular smooth muscle cells, this concentration of forskolin hyperpolarized the membrane and reduced the amplitude, frequency, duration and dV/dt_max of slow waves (Fig. 11A and C; Table 3). SIN1 (100 µM) hyperpolarized the membrane of ICC-MY, without altering the amplitude, frequency, duration and dV/dt_max of pacemaker potentials (Fig. 11B; Table 3). On the other hand, SIN1 (100 µM) hyperpolarized the membrane of circular smooth muscle cells, reduced the amplitude and dV/dt_max of slow waves, with no alteration in the frequency and duration of slow waves (Fig. 11D; Table 3). Thus the amplitude and dV/dt_max of slow waves were inhibited by the hyperpolarization with either forskolin or SIN1, while the amplitude and dV/dt_max of pacemaker potentials were not affected by the hyperpolarization. These results are consistent with the alteration found in the guinea-pig gastric antrum during hyperpolarization with K⁺ channel openers (Kito & Suzuki, 2003b), where hyperpolarization-induced inhibition largely affects the passive component of electrical activity. It is also found that cyclic nucleotide-related compounds do not inhibit pacemaker currents in intact tissues.

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Figure 11. Effects of forskolin and SIN1 on pacemaker potentials and slow waves recorded from mouse small intestine
A and B, pacemaker potentials recorded during application of 5 µM forskolin (A) or 100 µM SIN1 (B) (applied as indicated by the horizontal bars). C and D, slow waves recorded during application of 5 µM forskolin (C) or 100 µM SIN1 (D) (applied as indicated by the horizontal bars). The resting membrane potentials were: A -73 mV, B -69 mV, C -63 mV, D -64 mV. All traces were recorded from different tissues.

DISCUSSION

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Abstract
Introduction
Methods
Results
Discussion
References

In the mouse small intestine, slow waves and pacemaker potentials were recorded. Visualization of recorded cells by the infusion of Lucifer Yellow revealed that slow waves were generated in circular smooth muscle cells and pacemaker potentials were generated in ICC-MY. The results also indicated that Lucifer Yellow, infused into a single ICC-MY, spread in all planar directions more than 500 µm from the injected cell, as in the case of the guinea-pig small intestine (Belzer et al. 2002). It is interesting to note that the infused dye spread only to surrounding ICC and not to other types of cell including smooth muscle cells. One possible explanation for the spread of dye to particular cells in the tissue is that the conductivity of gap junctions to Lucifer Yellow is greater between ICC-MY than it is between ICC-MY and smooth muscle cells (Dickens et al. 1999). Alternatively, the electrical coupling between ICC-MY and circular smooth muscle cells might not be mediated by gap junctions, but by peg and socket junctions, which do not allow passage of dye (Thuneberg & Peters, 2001). In fact, it is reported that in small intestine, gap junctions between ICC-MY and smooth muscles are very rare or absent (Thuneberg et al. 1995; Daniel et al. 1998).

The present experiments indicated that in the mouse small intestine, pacemaker potentials recorded from ICC-MY have two components, a primary component and a plateau component. The primary component is selectively inhibited by NiCl₂, and is also inhibited in the absence of Ca²⁺ or during depolarization with high K⁺ solution. These results indicate that the primary component is generated by activation of voltage-dependent Ni²⁺-sensitive Ca²⁺-permeable channels. Since experiments were carried out in the presence of nifedipine (3 µM), involvement of voltage-dependent L-type Ca²⁺ channels may not be a key factor in initiating pacemaker potentials. It is considered that the voltage-dependent T-type Ca²⁺ channels, thought to be inhibited by low concentrations of Ni²⁺ (Perez-Reyes, 2003), are activated during the generation of the primary component. In cultured ICC-MY of mouse small intestine, a voltage-dependent dihydropyridine-resistant Ca²⁺ conductance is recorded (Kim et al. 2002), and the present experiments cannot rule out the possible involvement of this conductance. It is also possible that the voltage-dependent non-selective cation conductance recorded from mouse colonic smooth muscle cells (Koh et al. 2001) contributes to the generation of the primary component of pacemaker potentials. On the other hand, the plateau component of pacemaker potentials is inhibited by DIDS or low [Cl^-]_o solution, and also by application of nominally Ca²⁺-free solution. In addition, reduction of intracellular Ca²⁺ concentrations with BAPTA-AM abolished pacemaker potentials. These results suggest that the plateau component is generated by activation of Ca²⁺-activated Cl^- channels.

Unitary potentials were generated in the interval between pacemaker potentials, in the presence of nominally Ca²⁺-free solution or BAPTA-AM. These results raise the possibility that unitary potentials are generated by activation of Ca²⁺-inhibited non-selective cation channels (Koh et al. 2002). Alternatively, an instantaneous generation of a certain amount of unitary potentials is required to form pacemaker potential and impaired intracellular Ca²⁺ handlings cause the reduced occurrence of unitary potential generation, allowing visualization of individual events more frequently. However, the generation of unitary potentials was also facilitated during depolarization with high K⁺ solutions, as has been seen in gastric pacemaker cells of the guinea-pig (Kito et al. 2002c). Depolarization with high K⁺ solution will elevate intracellular Ca²⁺ concentrations (Fukuta et al. 2002), which may facilitate the generation of unitary potentials while the primary component is absent. Thus the cellular mechanism for the generation of unitary potentials in ICC-MY remains unclear. In the presence of NiCl₂, pacemaker potentials with only the plateau component are generated, while in the presence of DIDS, pacemaker potentials with the primary component alone are generated. Pacemaker potentials were abolished in the presence of both NiCl₂ and DIDS. Thus primary and plateau components are generated independently. These results also suggest that unitary potentials are not the essential factor in the generation of pacemaker potentials, unlike slow potentials generated in the circular muscle of the guinea-pig stomach (Edwards et al. 1999). Alternatively, the primary and plateau components are generated by different populations of unitary potentials. Taken together, these results suggest that the electro-physiological characteristics of pacemaker potentials generated in ICC-MY of mouse small intestine in situ are not identical to those of pacemaker activities recorded from cultured ICC-MY (Tokutomi et al. 1995; Koh et al. 1998; Thomsen et al. 1998; Huizinga et al. 2002; Nakayama & Torihashi, 2002).

Although we did not try simultaneous recordings of pacemaker potentials and slow waves with two microelectrodes, ICC-MY and circular smooth muscle cells seemed to be electrically coupled to each other, because both were generated with similar frequency and duration. However, the configuration of slow waves is distinct from that of pacemaker potentials. In slow waves, the primary component was inhibited by NiCl₂ and the plateau component was inhibited by DIDS or low [Cl^-]_o solution, as in the case of pacemaker potentials. Both the amplitude and dV/dt_max of slow waves were smaller than those of pacemaker potentials. Hyperpolarization of the membrane induced by forskolin or SIN1 decreased the amplitude and dV/dt_max of slow waves, with no alteration to those of pacemaker potentials. These results suggest that slow waves are formed passively by electrotonic spread of pacemaker potentials, as in the case of gastric antrum (Dickens et al. 1999; Hirst & Edwards, 2001; Kito & Suzuki, 2003a). However, it remains unclear how many parts of slow waves are formed by passive electrotonic conduction from pacemaker cells. It also remains unclear whether any active components produced by circular smooth muscle cells are involved in slow waves recorded in the mouse small intestine, as in the case of the second component of slow waves recorded from the guinea-pig gastric antrum (Dickens et al. 1999; Kito & Suzuki, 2003b).

Slow waves have a quick and transient repolarization component after the generation of the primary component, and the amplitude of this repolarization component was correlated with the dV/dt_max of slow waves. Since the dV/dt_max of slow waves decreased in nominally Ca²⁺-free solution (authors' unpublished observations), this component might be due to an influx of Ca²⁺. It is reasonable to speculate that the repolarization component is formed by activation of Ca²⁺-activated K⁺ channels, since slow waves are associated with an increased [Ca²⁺]_i in gastric muscles even in the presence of nifedipine (Fukuta et al. 2002). Alternatively, transient outward K⁺ current (I_to) may be activated during the initial depolarizing phase, as in the case of cardiac muscles (Sah et al. 2003). In fact, it has been reported that I_to is detected in isolated single smooth muscle cells of mouse small intestine (Lee et al. 2002). The transient repolarization component after the generation of the primary component has not been observed in pacemaker potentials, suggesting that it may be produced as an active response of circular smooth muscle membrane. A wide variation of dV/dt_max in slow waves was observed in the presence of tetrodotoxin (authors' unpublished observations), suggesting the absence of any neurogenic variation. As slow waves may be generated mostly by passive conduction, the variation seen in the dV/dt_max may be related to the fluctuation of the dV/dt_max occurring in pacemaker potentials. It remains unclear how these fluctuations arise in pacemaker potentials.

Coupling of IP₃-induced Ca²⁺ release from internal stores and mitochondrial activity may be involved in the generation of spontaneous activity in cultured ICC-MY of murine small intestine (Ward et al. 2000) or circular muscle tissues of the guinea-pig stomach antrum (Kito et al. 2002b). In the guinea-pig gastric antrum, pacemaker potentials are initiated by a burst of unitary potentials generated by the activation of Ca²⁺-activated Cl^- channels (Hirst et al. 2002a; Kito et al. 2002c). The present experiments indicated that BAPTA-AM abolishes rhythmic electrical activities of pacemaker cells. These results indicate that intracellular Ca²⁺ handling mechanisms may be involved in the generation of pacemaker potentials. In the mouse small intestine, inhibitors of Ca²⁺-ATPase at the internal stores (CPA and thapsigargin), inhibitors of IP₃-induced Ca²⁺ release (xestospongin C) and mitochondrial protonophores (FCCP and CCCP) inhibit the generation of slow waves (Ward et al. 2000; Malysz et al. 2001). However, the effects of these drugs include depolarization of the membrane. The present experiments indicated that depolarization of the membrane inhibits the primary component of pacemaker potentials. Therefore, the effects of these drugs on pacemaker potentials may require careful evaluation, although it would be important to investigate the precise role of Ca²⁺ release from IP₃-sensitive Ca²⁺ stores coupled with mitochondrial function in the generation of pacemaker potentials.

There are some similarities and some dissimilarities in pacemaker potentials recorded from guinea-pig gastric antrum compared with those recorded from mouse small intestine. The similar properties are as follows. (i) Pacemaker potentials consist of two components, primary and plateau components (Hirst & Edwards, 2001; Kito et al. 2002a; Kito & Suzuki 2003a). (ii) The primary component is generated by activation of voltage-dependent Ca²⁺-permeable channels, while the plateau component is generated by the opening of Ca²⁺-activated Cl^- channels (Hirst & Edwards, 2001; Kito et al. 2002a; Kito & Suzuki, 2003a). (iii) Unitary potentials are generated during the plateau component and the interval between pacemaker potentials (Hirst & Edwards, 2001; Kito et al. 2002c). (iv) Both the amplitude and dV/dt_max of pacemaker potentials are much larger than those of slow waves (Dickens et al. 1999, 2000; Kito & Suzuki 2003b). (v) BAPTA-AM abolishes pacemaker potentials (Kito et al. 2002a, c; Kito & Suzuki, 2003a). (vi) The amplitude of pacemaker potentials is not inhibited by hyperpolarization (Kito & Suzuki, 2003b). Thus the configuration of pacemaker potentials is quite similar between these two tissues.

On the other hand, there are two distinctions between the two types of pacemaker potentials. (i) The duration of pacemaker potentials is much longer in the stomach (7-10 s) than in the small intestine (about 1 s), and this may be related to the higher frequency of generation of pacemaker potentials in the small intestine (20-30 min^-1) than in the stomach (2-5 min^-1) (Hirst & Edwards, 2001; Kito et al. 2002a). (ii) Depolarization of the membrane with high K⁺ solution increases the frequency of pacemaker potentials in the stomach (Kito et al. 2002a), while it decreases the frequency in the small intestine (present experiments). Interestingly, the duration of pacemaker potentials is decreased during depolarization with high K⁺ solution in the guinea-pig gastric antrum (Kito et al. 2002a), while it is increased in the mouse small intestine.

In conclusion, the present study indicates that in the mouse small intestine, ICC-MY generate ongoing pacemaker potentials and circular smooth muscle cells generate slow waves in situ. Pacemaker potentials are formed by a primary component with an initial rapid rising potential, generated by activation of voltage-dependent Ca²⁺-permeable channels, and a following plateau component, generated by the opening of Ca²⁺-activated Cl^- channels. Slow waves may be generated mainly by passive electrotonic conduction of pacemaker potentials generated in ICC-MY. Some differences in the membrane properties are noted between intact and cultured ICC-MY.

REFERENCES

Top
Abstract
Introduction
Methods
Results
Discussion
References

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Acknowledgements

The authors are grateful to Dr Frank R. Edwards for critical reading of the manuscript and Dr Hiromichi Takano for technical advice. The present experiments were supported partly by a Grant-in-Aid for the Scientific Research (C) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (No. 14570044) and the Japan-Australia Research Cooperative Program to H. S.

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Belzer V, Kobilo T, Rich A & Hanani M (2002). Intercellular coupling among interstitial cells of Cajal in the guinea pig small intestine. Cell Tissue Res 307, 15-21	[Medline]
Daniel EE, Wang YF & Cayabyab F (1998). Role of gap junctions in structural arrangements of interstitial cells of Cajal and canine ileal smooth muscle. Am J Physiol 274, G1125-1141	[Medline]
Dickens EJ, Edwards FR & Hirst GDS (2000). Vagal inhibition in the antral region of guinea pig stomach. Am J Physiol 279, G388-399
Dickens EJ, Hirst GDS & Tomita T (1999). Identification of rhythmically active cells in guinea-pig stomach. J Physiol 514, 515-531	[Abstract/Full Text]
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