Propagation of slow waves requires IP3 receptors and mitochondrial Ca2+ uptake in canine colonic muscles

doi:10.1113/jphysiol.2003.040097

Propagation of slow waves requires IP₃ receptors and mitochondrial Ca²⁺ uptake in canine colonic muscles

Sean M. Ward, Salah A. Baker, Andrew de Faoite and Kenton M. Sanders

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557, USA

ABSTRACT

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Abstract
Introduction
Methods
Results
Discussion
References

In the gastrointestinal (GI) tract electrical slow waves yield oscillations in membrane potential that periodically increase the open probability of voltage-dependent Ca²⁺ channels and facilitate phasic contractions. Slow waves are generated by the interstitial cells of Cajal (ICC), and these events actively propagate through ICC networks within the walls of GI organs. The mechanism that entrains spontaneously active pacemaker sites throughout ICC networks to produce regenerative propagation of slow waves is unresolved. Agents that block inositol 1,4,5-trisphosphate (IP₃) receptors and mitochondrial Ca²⁺ uptake were tested on the generation of slow waves in the canine colon. A partitioned chamber apparatus was used to test the effects of blocking slow-wave generation on propagation. We found that active propagation occurred along strips of colonic muscle, but when the pacemaker mechanism was blocked in a portion of the tissue, slow waves decayed exponentially from the point where the pacemaker mechanism was inhibited. An IP₃ receptor inhibitor, mitochondrial inhibitors, low external Ca²⁺, and divalent cations (Mn²⁺ and Ni²⁺) caused exponential decay of the slow waves in regions of muscle exposed to these agents. These data demonstrate that the mechanism that initiates slow waves is reactivated from cell-to-cell during the propagation of slow waves. Voltage-dependent conductances present in smooth muscle cells are incapable of slow-wave regeneration. The data predict that partial loss of or disruptions to ICC networks observed in human motility disorders could lead to incomplete penetration of slow waves through GI organs and, thus, to defects in myogenic regulation.

(Resubmitted 24 January 2003; accepted after revision 4 March 2003; first published online 28 March 2003)

INTRODUCTION

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Abstract
Introduction
Methods
Results
Discussion
References

Electrical slow waves determine the frequency and amplitude of phasic contractions in gastrointestinal (GI) muscles (see Szurszewski, 1987). Slow waves originate in specialized pacemaker regions such as the border between the circular and longitudinal muscle layers in the stomach and small intestine and along the submucosal surface of the circular muscle layer in the colon (Sanders, 1996). Each pacemaker region is populated with interstitial cells of Cajal (ICC), and several studies have demonstrated the pacemaker role of these cells (e.g. Langton et al. 1989; Ward et al. 1994; Huizinga et al. 1995; Torihashi et al. 1995; Dickens et al. 1999; Ördög et al. 1999). ICC express specialized conductances, such as dihydropyridine-resistant Ca²⁺ conductances (Lee & Sanders, 1993; Kim et al. 2002b), a Ca²⁺-inhibited, non-selective cation conductance (Koh et al. 2002) and a Ca²⁺-activated Cl^- conductance (Hirst et al. 2002) that could contribute to the generation and propagation of slow waves. Slow waves spread electrotonically through low-resistance gap junctions and depolarize neighbouring smooth muscle cells (Dickens et al. 1999). Depolarization of smooth muscle cells activates voltage-dependent Ca²⁺ channels and facilitates Ca²⁺ entry and contraction (for review see Horowitz et al. 1999).

ICC generate spontaneous inward currents and slow-wave-like depolarizations (Koh et al. 1998; Thomsen et al. 1998). The spontaneous inward currents (pacemaker currents) of the murine small intestine are due mainly to periodic activation of non-selective cation channels. Activation of the pacemaker current involves complex Ca²⁺ handling, including periodic release of Ca²⁺ from the sarcoplasmic reticulum via inositol 1,4,5-trisphosphate (IP₃)-receptor-operated channels, uptake of Ca²⁺ by the mitochondria and reuptake of Ca²⁺ by the sarcoplasmic reticulum (Sanders et al. 2000; Ward et al. 2000). Blockade of any of these steps leads to the cessation of pacemaker currents in the ICC of the murine small intestinal .

ICC form extensive networks that run longitudinally and circumferentially within the tunica muscularis of the GI tract. ICC networks are thought to be critical for active propagation of slow waves. For example, dissection experiments have shown that slow waves decay exponentially in regions from which ICC networks have been removed (e.g. Sanders et al. 1990), and slow waves do not actively propagate through regions that are devoid of ICC due to pathological loss (Ördög et al. 1999). While many GI motility disorders have been shown to be associated with loss of ICC (see Sanders et al. 1999; Vanderwinden & Rumessen, 1999 for reviews), few show a complete lesion in this population of cells. Thus, the question of ICC propagation is important because it is possible that ICC remaining in abnormally functioning tissues may be capable of generating slow waves, and the real defect resulting from loss of ICC may be an inability to actively propagate these events through areas of organs normally serviced by phasic electrical activity. Without active propagation, the slow-wave mechanism would be rendered useless as a means of organizing phasic contractions in GI muscles, and important physiological behaviours, such as segmentation and gastric peristalsis would be disrupted.

How the active propagation of slow waves is accomplished is currently unresolved, but it is possible that the mechanism responsible for the generation of slow waves is sequentially activated from cell-to-cell such that slow waves are regenerated and spread without decrement over many centimetres of tissue. In the present study we tested whether drugs and conditions shown to block slow-wave generation in pacemaker cells can also affect the regeneration of slow waves. We tested whether an IP₃ receptor antagonist and mitochondrial inhibitors, which have been shown to inhibit slow-wave generation in murine small intestine (Ward et al. 2000), also block the generation of slow waves in colonic muscles. After establishing that the agents effectively blocked slow-wave generation, we used a partitioned chamber to determine the fate of colonic slow waves propagating into regions of muscle in which the slow-wave mechanism is inactivated. Canine colonic muscles were chosen for these studies because slow waves propagate with constant velocity and uniform waveform over many centimetres, and the long strips of muscle required for use in a partitioned chamber bath can be easily prepared (Sanders et al. 1990; Ward & Sanders, 1990).

METHODS

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Abstract
Introduction
Methods
Results
Discussion
References

Tissue preparation

Mongrel dogs of both sexes were killed with an intravenous injection of sodium pentobarbital (100 mg kg^-1) into the forelimb. The abdomen was opened and a segment of proximal colon, 8-12 cm from the ileocolonic sphincter, was removed. The colonic segment was opened along the mesenteric border and cleaned of faecal material with Krebs-Ringer bicarbonate solution (KRB). Sheets of proximal colon were pinned out in a dissecting dish and immersed in oxygenated KRB. Strips of tissue (1 mm times 50 mm) were cut parallel to the circular muscle fibres, and the mucosa was removed. The use and treatment of animals was approved by the Animal Use and Care Committee at the University of Nevada.

Electrophysiological recording

The strips of muscle were transferred to a recording chamber with a Sylgard silicone elastomer floor (Dow Corning, Midland, MI, USA) and pinned out in cross-section, allowing selective impalement of cells at any point through the tunica muscularis. Cells near the submucosal surface of the circular muscle layer were selectively impaled in this study because these cells are close to the source of pacemaker activity (Smith et al. 1987). Impalements of cells were made with glass microelectrodes having resistances of 50-90 M $Omega$ . Transmembrane potential was recorded with a standard electrometer (Intra 767; World Precision Instruments, Sarasota, Florida, USA). Data were recorded on digital tape (Vetter, Robersburg, PA, USA) and hard copies were made by replaying the tapes through a polygraph (Gould RS 3200, Cleveland OH, USA).

In some experiments the goal was to determine the effects of various agents on slow-wave propagation, and a partitioned recording chamber was used (Fig. 1). A recording dish was separated into two chambers by a thin strip of Plexiglas with a 5 mm hole drilled at the level of the Sylgard elastomer floor. A sheet of latex rubber was glued to one side of the Plexiglas partition, and a small hole was made in the latex to communicate through the partition. Muscles were pulled through the latex diaphragm and pinned to the Sylgard elastomer floor such that impalements of cells could be made in either chamber. The two chambers were perfused independently with warmed, oxygenated KRB. The latex partition sealed around the muscle strips, and solutions from either chamber did not leak into the adjacent chamber. This was tested by the addition of methylene blue dye to one chamber. The dye did not leak through to the adjacent chamber, and stained the muscle strip along a discrete line defined by the partition. In muscles in the partitioned bath, slow waves occurred spontaneously and propagated without decrement through both chambers (Fig. 1A and B). Dual electrode impalements of cells in both chambers showed a 1:1 coupling of spontaneous slow waves and propagation with constant latency of slow waves evoked by electrical field stimulation (EFS) in chamber A (Fig. 1C and D). Chamber A was continuously perfused with KRB throughout experiments to sustain spontaneous generation of slow waves within the portion of the muscle within this chamber. After control recordings to test the continuity of the muscle strip and the ability of slow waves to actively propagate through the strip, the solution perfusing chamber B was switched to a variety of test solutions with drugs that have been shown to block slow waves in open-bath experiments. Cells at various distances from the partition were impaled in chamber B to test the effects of these drugs on slow-wave propagation.

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Figure 1. Schematic diagram of the recording chamber used to study slow-wave propagation
A recording chamber was divided by a latex partition through which a strip of muscle was pulled and secured to the Sylgard elastomer base for recording. Chamber A was maintained as a slow-wave generation chamber and perfused with Krebs-Ringer bicarbonate solution (KRB). After control recordings, test solutions containing agents that inhibit the generation of slow waves were perfused through chamber B. Cells at various distances from the partition were impaled to test the effects of inhibiting the slow-wave mechanism on propagation. A and B, during control recordings, slow waves of approximately the same characteristics were recorded in both chambers. C and D, dual electrical recording proved that spontaneous slow waves propagated throughout the muscle, and there was a 1:1 correlation between the events recorded in the two chambers. This was also demonstrated by evoking slow waves in chamber A with electrical field stimulation (EFS, arrows). Evoked events were first recorded at the electrode in chamber A, and then they propagated with constant velocity to chamber B. Lines at the right of D highlight the latencies between slow waves recorded in the two chambers.

Solutions and drugs

Tissues were constantly perfused with oxygenated KRB of the following composition (mM): NaCl 118.5, KCl 4.5, MgCl₂ 1.2, NaHCO₃ 23.8, KH₂PO₄ 1.2, dextrose 11.0 and CaCl₂ 2.4, pH 7.4 at 37 ± 0.5 °C. The pH of the KRB was 7.3-7.4 when bubbled with 95 % O₂-5 % CO₂. After pinning, the muscles were left to equilibrate for at least 2 h before experiments were begun. 2-aminoethyl diphenyl borate (2-APB), antimycin A, carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), carbonyl cyanide m-chlorophenylhydrazone (CCCP), tetrodotoxin, oligomycin, nifedipine, nickel chloride and manganese chloride were all obtained from Sigma (St Louis, MI, USA). 2-APB, tetrodotoxin, nickel and manganese were dissolved in dH₂O; antimycin A, FCCP, oligomycin and nifedipine were all dissolved in absolute ethanol; CCCP was dissolved in DMSO. Further dilutions of each drug were performed with the superfusion buffer to the concentration stated in the text.

Analysis of intracellular microelectrode data

Data are expressed as the mean ± S.E.M. Differences in the data were evaluated by Student's unpaired t test. The level of statistical significance was taken as P < 0.05. The 'n' values reported in the text refer to the number of tissue strips from which recordings were performed, with each muscle strip that was exposed to the same drug being taken from a separate animal. Several slow-wave parameters were analysed. (1) Resting membrane potential (RMP). (2) Slow-wave amplitude. (3) Duration (time from 10 % maximum depolarization to 90 % repolarization) and (4) frequency. The conduction velocity between two electrodes was also measured in some experiments. Slow waves were evoked at the end of the muscle strip in these studies, and the distance was measured between points of recording. Conduction velocity (CV) was calculated as the distance between recording sites divided by the latency between the times of 10 % of maximal slow-wave depolarization at each recording site. Figures displayed were made from digitized data using Adobe Photoshop 4.0.1 (Adobe, Mountain View, CA, USA) and Corel Draw 7.0 (Corel, Ontario, Canada).

RESULTS

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Abstract
Introduction
Methods
Results
Discussion
References

Spontaneous electrical slow waves were recorded from smooth muscle cells along the submucosal surface of the circular muscle layer. RMPs in this region averaged -78 ± 2 mV, and slow waves of 38.0 ± 3 mV in amplitude and 4.2 ± 0.2 s in duration occurred at a frequency of 5.5 ± 0.5 cycles min^-1 (n = 12). These events were consistent in waveform and frequency to previously published recordings from canine colonic muscles (Smith et al. 1987).

The importance of IP₃-receptor-operated stores on slow-wave generation has been demonstrated in murine GI muscles and ICC (Suzuki et al. 2000; Ward et al. 2000). We tested whether colonic slow-wave generation also depends upon IP₃-receptor-operated Ca²⁺ release by evaluating the effects of 2-APB, an inhibitor of IP₃-dependent Ca²⁺ release. 2-APB (30, 60 and 90 µM) caused concentration-dependent depolarization of membrane potential and inhibition of slow waves. In these experiments, the RMP of colonic muscle cells averaged -79.8 ± 2.7 mV, and slow waves of 36.7 ± 3.1 mV in amplitude and 4.3 ± 0.3 s in duration occurred at a frequency of 5.2 ± 0.4 cycles min^-1 (n = 6; Fig. 2A). Addition of 2-APB (30 µM) caused depolarization to -68 ± 8.4 mV and a reduction in slow-wave amplitude, duration and frequency to 12.6 ± 4.7 mV (P < 0.005), 3.0 ± 1.5 s (P < 0.05) and 2.0 ± 1.2 cycles min^-1 (P < 0.01; Fig. 2B), respectively. Increasing 2-APB (60 µM) further depolarized the membrane potential to -55.5 ± 4.8 mV and inhibited slow waves (n = 4; P < 0.01). Blocking of slow waves was associated with an increase in electrical noise (see Fig. 2C). In one experiment, a higher concentration of 2-APB (90 µM) was tested. This blocked slow waves within 5 min, and caused a marked depolarization in membrane potential of nearly 40 mV (Fig. 2D).

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Figure 2. Effects of 2-APB on slow waves
A, control slow waves recorded in KRB solution. B, electrical activity after the introduction of 30 µM 2-APB; slow waves were greatly reduced in amplitude and irregular in frequency. C, inhibition of slow waves in the presence of 60 µM 2-APB. Only random noise was resolvable in the traces. D, after 90 µM 2-APB, all activity was inhibited. Note the increasing level of depolarization associated with exposure to 2-APB. Previous studies have shown, however, that depolarization to -63 mV, as with 2-APB at 60 µM in this example, does not block slow waves in the canine colon (Ward et al. 1990).

Mitochondrial Ca²⁺ uptake has been suggested to be of fundamental importance to the generation of slow waves in murine GI muscles (Ward et al. 2000). We tested the role of mitochondria in the generation of slow waves in canine colonic muscles using mitochondrial inhibitors. Antimycin A, an inhibitor of complex III of the electron transport chain causes collapse of the mitochondrial membrane potential and subsequent inability of the mitochondria to sequester Ca²⁺ from the cytoplasm (Schinder et al. 1999; Tinel et al. 1999). In these experiments resting membrane potential (RMP) averaged -78 ± 1.5 mV, and slow waves 35.8 ± 1.7 mV in amplitude occurred at a frequency of 6.4 ± 0.24 cycles min^-1. Addition of antimycin A (10 µM for 6 min) caused a decrease in slow-wave amplitude and frequency to 9.2 ± 5.7 mV and 2 ± 1.2 cycles min^-1, respectively (P < 0.01 for both parameters, n = 6). After 8 min, slow waves were completely abolished without significant change in membrane potential (i.e. -74 ± 2.3 mV, P > 0.05; Fig. 3).

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Figure 3. Effects of antimycin A on slow-wave generation
A, control slow-wave activity before effects of antimycin A (10 µM added at the black arrow). B, 5 min after the addition of antimycin A, slow-wave frequency and amplitude were significantly depressed. C, after 8 min, slow waves were completely inhibited. D and E, time dependence of the effects of antimycin A on slow-wave amplitude and frequency, respectively (n = 6). The times for half-inhibition of slow-wave amplitude and frequency were 5.39 and 5.43 min, respectively.

We also investigated the effects of the protonophores FCCP and CCCP on the generation of slow waves. These compounds collapse the proton gradient across the inner mitochondrial membrane, decreasing the driving force for Ca²⁺ uptake (Farkas et al. 1989). In these experiments, RMP averaged -77.0 ± 1.0 mV, and slow waves 38 ± 1.8 mV in amplitude and 7.2 ± 1.0 s in duration occurred at a frequency of 4.9 ± 1.6 cycles min^-1 (n = 15). Following the addition of FCCP (1 µM), the membrane potential depolarized to -67.0 ± 1.6 mV and slow waves were completely inhibited within 10 min (P < 0.05). CCCP produced similar effects. In this series of experiments, RMP averaged -78.0 ± 1.5 mV, and slow waves 38 ± 2 mV in amplitude occurred at a frequency of 5.8 ± 0.5 cycles min^-1 (n = 6). CCCP (1 µM) depolarized the membrane potential to -69.0 ± 2.0 mV (P < 0.05) and inhibited slow waves within 10 min (Fig. 4). The effects of protonophores were not affected by tetrodotoxin (0.3 µM; n = 3; data not shown).

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Figure 4. Effects of CCCP on slow-wave generation
A, effects on slow-wave activity of 1 µM CCCP, which was applied as indicated by the black arrow. B, 6 min after the addition of CCCP, slow-wave frequency and amplitude were significantly depressed. C, after 10 min, slow waves were completely inhibited. D and E, time dependence of the effects of CCCP on slow-wave amplitude and frequency, respectively (n = 6). The times for half inhibition of slow-wave amplitude and frequency were 6.80 and 6.48 min, respectively.

Since the mitochondrial agents tested might disrupt the production of ATP by oxidative metabolism, we tested a mitochondrial poison that is not known to affect mitochondrial Ca²⁺ uptake. Oligomycin blocks ATP synthesis by inhibiting the F₀/F₁ ATPase, and 10 µM of oligomycin for up to 20 min had no effect on membrane potential or the amplitude, duration or frequency of slow waves (data not shown). FCCP or CCCP added in the presence of oligomycin (10 µM) caused inhibition of electrical slow waves over the same time period as observed when these agents were applied alone (n = 5). These data suggest that the effects of FCCP and CCCP on slow waves are independent of the effects on ATP production.

Role of slow-wave generation mechanisms in slow-wave propagation

The data presented above are consistent with the model derived from studies of ICC from murine small intestine in which slow waves depend upon Ca²⁺ release from IP₃-receptor-operated stores and Ca²⁺ uptake by mitochondria. It has been shown previously that slow waves are also inhibited by reduced external Ca²⁺ (i.e. to 0.1 mM) and non-selective blockers of Ca²⁺ channels, such as Ni²⁺ and Mn²⁺ (Huizinga et al. 1991; Ward & Sanders, 1992). We tested whether the mechanisms involved in initiating slow waves were also required for slow-wave propagation, using a partition recording chamber (see Methods). In experiments utilizing partitioned chambers, RMPs and electrical slow-wave parameters were similar on both sides of the latex partition and not statistically different from those stated above (P > 0.05 for all parameters; Fig. 1B and C). Simultaneous electrical recordings from both chambers demonstrated that spontaneous slow waves originating from sites within the strip propagated throughout. This was also demonstrated by evoking slow waves with EFS (single pulses, 0.5-2 ms in duration) in chamber A and recording events with electrodes in both chambers with a constant latency between events. From the latencies we calculated that slow waves propagated from chamber A to chamber B with a conduction velocity of 16.5 ± 1.5 mm s^-1 (n = 15). This value confirms previous measurements of conduction velocity from the canine proximal colon (i.e. 17 mm s^-1; Sanders et al. 1990). To investigate the mechanisms affecting the propagation of slow waves, experiments were performed in which the KRB solution in chamber B was replaced with solutions shown to block slow-wave generation.

In the first experiment we tested the effects of reduced external Ca²⁺ at a concentration that has previously been shown to block colonic slow waves (Ward & Sanders, 1992). In these experiments, RMP averaged -73.4 ± 1.9 mV under control conditions. Slow waves with amplitudes of 29 ± 1.7 mV and durations of 4.4 ± 0.4 s occurred at a frequency of 7.0 ± 0.3 cycles min^-1 (n = 5). Introduction of low-Ca²⁺ KRB to chamber B caused a reduction in slow-wave amplitude that varied as a function of distance from the latex partition. For example, at 0.1 mm from the partition, RMP averaged -45.0 ± 2.1 mV and slow-wave amplitude was 7.1 ± 2.0 mV. Impalements of cells at various points from the partition showed that no activity could be recorded at distances greater than 4 mm from the partition. A plot of slow-wave amplitude as a function of distance from the partition was fitted with an exponential function with a length constant of 1.53 mm (R² = 0.9722; Fig. 5).

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Figure 5. Effects of reduced extracellular Ca²⁺ on slow-wave propagation
After recording control slow waves (A), the solution in chamber B was switched to KRB with 0.1 mM Ca²⁺. This concentration inhibits slow-wave generation in canine colonic muscles (Ward & Sanders, 1992). Cells were impaled at various distances from the latex partition in chamber B. Excerpts of records from each recording site are shown in B-G. There was a progressive decrease in slow-wave amplitude as a function of distance from the latex partition. The data from five experiments are summarized in H. Data points were fitted with an exponential function (see text for details).

General Ca²⁺ channel blockers, Ni²⁺ and Mn²⁺ also inhibited the active propagation of slow waves. In experiments testing the effects of Ni²⁺, RMP averaged -74 ± 3.1 mV, and slow waves 38.0 ± 6 mV in amplitude and 4.8 ± 0.3 s in duration occurred at a frequency of 6.3 ± 0.3 cycles min^-1 (n = 5). Fifteen minutes after the addition of Ni²⁺ (1 mM) to chamber B, slow-wave activity decreased in amplitude as a function of distance from the partition. For example, in cells 0.1 mm from the partition, the RMP averaged -70.0 ± 0 mV, and slow waves 9.7 ± 1.5 mV in amplitude (P < 0.01 compared to control activity) and 4.5 ± 0.3 s in duration occurred at a frequency of 6.3 ± 0.3 cycles min^-1. Recordings made from cells at various distances from the partition revealed a decay in slow-wave amplitude with distance (Fig. 6). Amplitude was plotted as a function of distance and fitted with an exponential function (R² = 0.9918) with a length constant of 0.53 mm (n = 5). In experiments testing Mn²⁺, the RMP averaged -76.0 ± 2.9 mV, and slow waves 37.0 ± 2.5 mV in amplitude and 4.4 ± 0.8 s in duration occurred at a frequency of 5.8 ± 0.7 cycles min^-1 under control conditions (n = 5). After addition of Mn²⁺ (1 mM) to chamber B, slow waves were reduced as a function of distance from the partition (Fig. 7). For example, cells at 0.25 mm from the partition had a resting potential of -65 ± 2.5 mV (P < 0.05 compared with control), and slow waves 15.2 ± 5 mV in amplitude and 3.4 ± 1.1 s in duration occurred at a frequency that was the same as during control recording (5.5 ± 0.8 cycles min^-1). Impalement of cells at various distances from the partition showed that slow waves decayed with distance. The amplitudes of slow waves were plotted and fitted with an exponential function (R² = 0.9853) with a length constant of 0.57 mm.

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Figure 6. Effects of Ni²⁺ on slow-wave propagation
After recording control slow waves (A), the solution in chamber B was switched to KRB with Ni²⁺ (1.0 mM). This concentration of Ni²⁺ abolishes slow waves in canine colonic muscles (Ward & Sanders, 1992). Cells were impaled at various distances from the latex partition in chamber B. Excerpts of records from each recording site are shown in B-E. There was a progressive decrease in slow-wave amplitude as a function of distance from the latex partition. The data from five experiments are summarized in F. Data points were fitted with an exponential function (see text for details).

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Figure 7. Effects of Mn²⁺ on slow-wave propagation
After recording control slow waves (A), the solution in chamber B was switched to KRB with Mn²⁺ (1.0 mM). This concentration of Mn²⁺ abolishes slow waves in canine colonic muscles (Ward & Sanders, 1992). Cells were impaled at various distances from the latex partition in chamber B. Excerpts of records from each recording site are shown in B-F. There was a progressive decrease in slow-wave amplitude as a function of distance from the latex partition. The data from five experiments are summarized in G. Data points were fitted with an exponential function (see text for details).

The effects of 2-APB on slow-wave propagation were studied in the next experiments. In these tests RMP averaged -78.0 ± 2.0 mV, and slow waves 36.0 ± 2.5 mV in amplitude and 4.5 ± 0.5 s in duration occurred at a frequency of 5.5 ± 0.5 cycles min^-1 (n = 5). After exposure to 2-APB (30 µM), RMP depolarized to -65.0 ± 5.0 mV and slow waves were abolished at distances greater than 2 mm from the partition (Fig. 8). Impalements of cells at various distances revealed that slow waves decayed in an exponential manner from the partition. At 0.5 mm from the partition, slow waves averaged 8.0 ± 1.3 mV in amplitude and decayed with a length constant of 0.53 mm (Fig. 8; R² = 0.9923).

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Figure 8. Effects of 2-APB on slow-wave propagation
After recording control slow waves (A), the solution in chamber B was switched to KRB with 2-APB (30 µM). Cells were impaled at various distances from the latex partition in chamber B. Excerpts of records from each recording site are shown in B-E. There was a progressive decrease in slow-wave amplitude as a function of distance from the latex partition. The data from five experiments are summarized in F. Data points were fitted with an exponential function.

Since slow waves were also abolished with mitochondrial inhibitors, we investigated the effects of adding antimycin A and protonophores to chamber B. In these tests, RMP averaged -78 ± 1.2 mV and slow waves 44.4 ± 3.3 mV in amplitude and 13.6 ± 2.1 s in duration occurred at a frequency of 4.0 ± 0.5 cycles min^-1 (n = 6). After 15 min exposure to antimycin A to inhibit intrinsic pacemaker activity at the site of recording, RMP had depolarized to -72 ± 2.5 (P > 0.05 compared with control), and slow waves 12.4 ± 2.5 mV in amplitude occurred at a frequency of 4.0 ± 0.25 cycles min^-1, 1.0 mm from the partition. Impalements of cells at various distances showed that slow waves decayed in an exponential manner from the partition with a length constant of 1.53 mm. Slow waves were not recorded at distances greater than 5 mm from the partition (Fig. 9; R² = 0.9904).

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Figure 9. Effects of antimycin A on slow-wave propagation
After recording control slow waves (A), the solution in chamber B was switched to KRB with antimycin A (10 µM). Cells were impaled at various distances from the latex partition in chamber B. Excerpts of records from each recording site are shown in B-G. There was a progressive decrease in slow-wave amplitude as a function of distance from the latex partition. The data from six experiments are summarized in H. Data points were fitted with an exponential function with a length constant of 1.53 mm and R² = 0.9904 (P < 0. 05).

Prior to the addition of FCCP, the RMP averaged -77 ± 0.6 mV, and slow waves 38 ± 1.8 mV in amplitude and 7.2 ± 0.9 s in duration occurred at a frequency of 5.8 ± 0.6 cycles min^-1 (n = 5). FCCP (after 15 min) added to chamber B produced a depolarization in RMP to -69.0 ± 2.0 mV (P < 0.05 compared with control) and a reduction in slow-wave amplitude to 30.5 ± 2.5 mV in cells along the submucosal surface of the circular muscle layer located immediately adjacent to the partition site on the control side. On the treated side of the chamber, RMP remained relatively constant such that at a site 4 mm from the partition it was -63 ± 0.7 mV (n = 5). Slow waves decayed exponentially from the partition, such that at 0.2 mm they were 10.6 ± 0.7 mV in amplitude and occurred at a frequency of 5.2 ± 0.6 cycles min^-1 (P > 0.05 compared with control). At a distance of 4 mm from the partition, slow waves were completely abolished. The length constant for the decay of slow-wave amplitude was 0.80 mm (Fig. 10; R² = 0.9964).

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Figure 10. Effects of FCCPon slow-wave propagation
After recording control slow waves (A), the solution in chamber B was switched to KRB with FCCP (1 µM). Cells were impaled at various distances from the latex partition in chamber B. Excerpts of records from each recording site are shown in B-F. There was a progressive decrease in slow-wave amplitude as a function of distance from the latex partition. The data from five experiments are summarized in G. Data points were fitted with an exponential function with a length constant of 0.8 mm and R² = 0.9964; (P < 0.05).

DISCUSSION

Top
Abstract
Introduction
Methods
Results
Discussion
References

In this study we found that the mechanisms responsible for slow-wave generation in murine pacemaker ICC are also probably responsible for the generation of slow waves in the canine proximal colon. This conclusion is supported by the observations that agents known to inhibit Ca²⁺ release from IP₃ receptors and mitochondrial Ca²⁺ uptake blocked slow-wave activity. Previous studies have shown that reduced external Ca²⁺ and cations that block Ca²⁺ channels, such as Ni²⁺ and Mn²⁺, also inhibit slow-wave generation (Ward et al. 1992) and these observations are consistent with the model for pacemaker activity in ICC (Ward et al. 2000). We also found that external Ca²⁺, IP₃-receptor-operated Ca²⁺ release and mitochondrial Ca²⁺ handling are required for the regeneration (active propagation) of slow waves. This was shown in strips in which slow-wave generation was preserved on one side of a latex partition, but active propagation of slow waves was blocked in another portion of the muscle by exposure to drugs known to reduce Ca²⁺ entry and inhibit IP₃-receptor-operated stores or mitochondrial Ca²⁺ uptake. These are important findings because they demonstrate for the first time that the unique pacemaker mechanism in ICC that is responsible for initiating slow waves is also required for regenerative propagation.

Previous studies have predicted that ICC might be necessary for the propagation of slow waves. Slow waves generated in ICC have been shown to spread passively to circular smooth-muscle cells in the guinea-pig gastric antrum (Dickens et al. 1999). Many studies in the past have been performed on isolated GI smooth-muscle cells, and these have failed to demonstrate a pacemaker mechanism or the appropriate conductances in smooth-muscle cells that could be responsible for electrical-slow waves. From these negative observations, we proposed that slow waves propagate in a regenerative manner within ICC networks, but conduct passively into smooth-muscle cells (see Horowitz et al. 1999). Removal of thin strips of muscle containing the slow-wave pacemaker cells in the canine proximal colon caused loss of regenerative spread of slow waves into adjacent smooth muscle tissue (Sanders et al. 1990). When ICC were reduced in number in murine gastric tissues by blocking receptor tyrosine kinase signalling, slow waves could still be recorded from areas containing ICC networks, but this activity did not spread to areas devoid of ICC (Ördög et al. 1999). These observations show that ICC are coupled to smooth muscles and suggest that without intact ICC networks, slow-wave activity does not regenerate and decays passively in the smooth-muscle syncytium. The present study demonstrates that even in the continued presence of ICC, inhibition of the pacemaker mechanism in a region of muscle blocks the active propagation of slow waves in that region. Thus, sequential, cell-to-cell reactivation of the pacemaker mechanism is necessary for the active propagation of slow waves, and the voltage-dependent conductances expressed by smooth-muscle cells are not capable of sustaining active propagation.

ICC from the stomach (Dickens et al. 1999; Kim et al. 2002a), small bowel (Koh et al. 1998; Thomsen et al. 1998; Ward et al. 2000) and colon (Langton et al. 1989) have intrinsic electrical rhythmicity. These cells are organized into electrically coupled networks and any cell within the network could conceivably initiate a pacemaker event. The dominant pacemaker site, event-to-event, is the first cell to generate sufficient inward current to entrain the other cells in the network. In intact muscles this 'primary pacemaker site' is unstable and multi-site recordings coupled with triangulation techniques have shown that dominance of a given site as the primary pacemaker is ephemeral (Publicover & Sanders, 1984). Once an inward current is initiated in a primary pacemaker site, it is not entirely clear how pacemaker units in coupled ICC are entrained in regenerative propagation. Rates of slow-wave propagation measured in intact muscles show that simple diffusion of Ca²⁺ or a second-messenger substance from cell-to-cell is too slow to be the primary factor entraining slow-wave pacemakers. To accomplish the propagation rates observed, it is highly likely that slow-wave propagation depends upon an electrical mechanism. ICC express dihydropyridine-insensitive Ca²⁺ channels (Lee & Sanders, 1993; Kim et al. 2002b), and it is possible that entrainment could result from voltage-dependent Ca²⁺ entry via such a conductance. Small increases in local Ca²⁺ near IP₃ receptors may initiate Ca²⁺ release (see Discussion in Kim et al. 2002b). Others have suggested that production of IP₃ is stimulated by depolarization, and this could be the entraining signal (Nose et al. 2000). The present study does not resolve the signal that entrains pacemaker activity in networks of ICC, but the data are consistent with the idea that regeneration requires the same mechanism that is responsible for generation of the primary pacemaker event. In other words, the mediator that entrains slow waves sequentially reactivates the local pacemaker mechanism as slow waves spread through the ICC networks.

We used the partitioned apparatus to test the effects of various compounds on slow-wave propagation because if a compound blocks slow waves in open-bath experiments, it is unclear whether the block comes from inhibition of slow-wave generation or from effects on propagation. There are probably some unavoidable technical problems with the partitioned chamber technique related to the diffusional mixing of solutions perfusing the two chambers within the interstitial spaces of the muscle strips. For example, our data showed exponential decay in slow waves as a function of distance from the partition within recording chamber B when agents were introduced that inhibit slow-wave generation. The fact that slow waves decay over a distance of millimetres from the partition suggests an electrical mechanism of conduction rather than a purely diffusional mechanism. Decay of slow waves over the distances noted would be predicted if active slow-wave propagation was blocked and slow waves were conducted passively through the smooth-muscle syncytium. Measurements of cable properties of GI muscles have reported length constants of the order of 1-2 mm (e.g. Abe & Tomita, 1968). The length constants measured from slow-wave decay in the present experiments are probably underestimations of the true length constants obtained in experiments in which square pulses of current are injected into smooth muscle syncytia. In our experiments it is unlikely that the site of inhibition of slow waves was discrete (i.e. exactly at the latex partition), and the concentrations due to diffusion or effectiveness of the agents added to chamber B in inhibiting slow waves may have varied somewhat within the smooth-muscle strips penetrating into chamber A. Thus, in some cases we may have observed longer length constants if a particular agent diffused poorly into the muscle in chamber A, and in other cases slow waves may have been inhibited before the edge of partition and length constants in these instances would tend to be reduced. Since we had no intention of using this apparatus to precisely measure cable properties, these artefacts were acceptable.

It should also be noted that some of the test solutions added to chamber B that were associated with loss of slow-wave propagation also caused depolarization of the membrane potential. Thus, the question could be raised as to whether the test solutions blocked slow-wave propagation as a result of depolarization. We have reported previously that depolarization of canine proximal colon muscles to -50 mV (by adjusting external K⁺ concentration) reduced slow-wave amplitude to approximately 40 %, but did not block activity (Ward et al. 1990). Blockade of slow-wave propagation by most of the test conditions in the present study was associated with smaller depolarizations; however, reduced external Ca²⁺ (0.1 mM) depolarized tissues to an average of -45 mV. At this membrane potential our previous study predicts that slow waves would be reduced in amplitude by the compounds used in the present study, but not blocked.

Morphological studies have shown that the organization of pacemaker cells in the GI tract is quite different from the heart, where pacemaker cells are localized in nodal tissues. After the initiation of electrical activity in nodal cells of the heart, it spreads to the myocardium where it is regenerated by cardiac muscle cells via voltage-dependent conductances. The present study demonstrates how a nodal organization of pacemaker cells would not be suitable in the gut due to the necessity of sequential activation of the pacemaker mechanism in ICC for regenerative propagation and the inability of smooth muscle cells to regenerate slow waves. Pacemaker ICC in GI muscles form extensive networks within the tunica muscularis and the networks of ICC are distributed around the circumference of the GI organs and along the length of the phasic regions of the gut (Thuneberg, 1982; Rumessen & Thuneberg, 1996; Burns et al. 1997). Such a broad distribution of pacemaker cells is consistent with the notion that these cells provide a pathway for active propagation. The problem of slow-wave distribution is compounded in the thicker-walled GI organs of large animals where ICC networks within a single plane of the tunica muscularis might not be capable of generating enough current to activate all the muscle cells. A recent study of the canine gastric antrum demonstrated that in thicker-walled GI organs, pacemaker ICC, which are capable of generating and regenerating slow waves, are distributed throughout the tunica muscularis (Horiguichi et al. 2001). ICC that line muscle bundles in these tissues probably provide the active propagation pathway necessary to carry slow waves through the thickness of the muscle layers.

Studies of human tissues have revealed that ICC are lost or reduced in a variety of motility disorders (see Sanders et al. 1999; Vanderwinden & Rumessen, 1999 for reviews). Total loss of ICC would be extremely detrimental to motility because both pacemaker function and inputs from the enteric nervous system would be impaired. Most cases documenting lesions in ICC networks, however, have shown reductions or heterogeneous distributions of ICC rather than a complete loss. The present findings, therefore, have important implications for such cases because the major defects in motility might arise from inappropriate propagation of slow-wave activity rather than a total loss of electrical rhythmicity. In cases where ICC are partially lost, slow waves may be generated in regions with intact ICC, and this activity might be recorded with extracellular electrodes or electrogastrogram techniques. These data suggest that a given motility problem is not associated with defects in electrical rhythmicity. Our data would predict that active propagation of slow waves would be blocked in regions where ICC networks are disrupted. Smooth muscle cells would be unable to sustain regeneration of slow waves because these cells lack the basic mechanism required to actively propagate slow waves. Thus, motility dysfunction might result from structural defects in which the continuity of the ICC networks is disrupted. More extensive studies in which the integrity of slow-wave propagation is evaluated may be necessary to relate functional defects to abnormalities in ICC networks.

REFERENCES

Top
Abstract
Introduction
Methods
Results
Discussion
References

Abe Y & Tomita T (1968). Cable properties of smooth muscle. J Physiol 196, 87-100 [Medline]

Burns AJ, Herbert TM, Ward SM & Sanders KM (1997). Interstitial cells of Cajal in the guinea-pig gastrointestinal tract as revealed by c-Kit immunohistochemistry. Cell Tiss Res 290, 11-20

Dickens EJ, Hirst GD & Tomita T (1999). Identification of rhythmically active cells in guinea-pig stomach. J Physiol 514, 515-531 [Abstract/Full Text]

Farkas DL, Wei M-D, Febbroriello P, Carson JH & Loew LM (1989). Simultaneous imaging of cell and mitochondrial membrane potential. Biophys J 56, 1053-1069 [Abstract]

Hirst GD, Bramich NJ, Teramoto N, Suzuki H & Edwards FR (2002). Regenerative component of slow waves in the guinea-pig gastric antrum involves a delayed increase in [Ca²⁺]_i and Cl^- channels. J Physiol 540, 907-919 [Abstract/Full Text]

Horiguchi K, Semple GSA, Sanders KM & Ward SM (2001). Distribution of pacemaker function through the tunica muscularis of the canine gastric antrum. J Physiol 537, 237-250 [Abstract/Full Text]

Horowitz BM, Ward SM & Sanders KM (1999). Cellular and molecular basis for electrical rhythmicity in gastrointestinal muscles. Ann Rev Physiol 61, 19-43 [Abstract/Full Text]

Huizinga JD, Farraway L & Den Hertog A (1991). Generation of slow-wave-type action potentials in canine colon smooth muscle involves a non-L-type Ca²⁺ conductance. J Physiol 442, 15-29 [Abstract]

Huizinga JD, Thuneberg L, Kluppel M, Malysz J, Mikkelsen HB & Bernstein A (1995). W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity. Nature 373, 347-349 [Medline]

Kim TW, Beckett EAH, Hanna R, Koh SDÖrdög T, Ward SM & Sanders KM (2002a). Regulation of pacemaker frequency in the murine gastric antrum. J Physiol 538, 145-157 [Abstract/Full Text]

Kim YC, Koh SD & Sanders KM (2002b). Voltage-dependent inward currents of interstitial cells of Cajal from murine colon and small intestine. J Physiol 541, 797-810 [Abstract/Full Text]

Koh SD, Jun JY, Kim TW & Sanders KM (2002). A Ca²⁺-inhibited non-selective cation conductance contributes to pacemaker currents in cultured interstitial cells of Cajal. J Physiol 540, 803-814 [Abstract/Full Text]

Koh SD, Sanders KM & Ward SM (1998). Spontaneous electrical rhythmicity in cultured interstitial cells of Cajal from the murine small intestine. J Physiol 513, 203-213 [Abstract/Full Text]

Langton P, Ward SM, Carl A, Norell MA & Sanders KM (1989). Spontaneous electrical activity of interstitial cells of Cajal isolated from canine proximal colon. Proc Natl Acad Sci U S A 86, 7280-7284 [Medline]

Lee HK & Sanders KM (1993). Comparison of ionic currents from interstitial cells and smooth muscle cells of canine colon. J Physiol 460, 135-152 [Abstract]

Nose K, Suzuki H & Kannan H (2000). Voltage dependency of the frequency of slow waves in antrum smooth muscle of the guinea-pig stomach. Jpn J Physiol 50, 625-633 [Medline]

Ördög T, Ward SM & Sanders KM (1999). Interstitial cells of Cajal generate electrical slow waves in the murine stomach. J Physiol 518, 257-269 [Abstract/Full Text]

Publicover NG & Sanders KM (1984). A technique to locate the pacemaker in smooth muscles. J Appl Physiol 57, 1586-1590 [Abstract]

Rumessen JJ & Thuneberg L (1996). Pacemaker cells in the gastrointestinal tract: interstitial cells of Cajal. Scand J Gastroenterol 31, 82-94

Sanders KM, (1996). A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract. Gastroenterology 111, 492-515 [Medline]

Sanders KMÖrdög T, Koh SD, Torihashi S & Ward SM (1999). Development and plasticity of interstitial cells of Cajal. Neurogastroenterol Motil 11, 311-338 [Medline]

Sanders KMÖrdög T, Koh SD & Ward SM (2000). A novel pacemaker mechanism drives gastrointestinal rhythmicity. News Physiol Sci 15, 291-298 [Abstract/Full Text]

Sanders KM, Stevens R, Burke E & Ward SM (1990). Slow waves actively propagate at submucosal surface of circular layer in canine colon. Am J Physiol 259, G258-263 [Medline]

Schinder AF, Olson EC, Spitzer NC & Montal M (1999). Mitochondrial dysfunction is a primary event in glutamate neurotoxicity. J Neurosci 16, 6125-6133 [Abstract/Full Text]

Smith TK, Reed JB & Sanders KM (1987). Origin and propagation of electrical slow waves in circular muscle of the canine proximal colon. Am J Physiol 252, C215-224 [Medline]

Suzuki H, Takano H, Yamamoto Y, Komuro T, Saito M, Kato K & Mikoshiba K (2000). Properties of gastric smooth muscles obtained from mice which lack inositol trisphosphate receptor. J Physiol 525, 105-111 [Abstract/Full Text]

Szurszewski JH, (1987). Electrical basis for gastrointestinal motility. In Physiology of the Gastrointestinal Tract, 2nd edn, ed. Johnson LR, pp. 383-422. Raven, New York

Thomsen L, Robinson TL, Lee JC, Farraway LA, Hughes MJ, Andrews DW & Huizinga JD (1998). Interstitial cells of Cajal generate a rhythmic pacemaker current. Nat Med 4, 848-851 [Medline]

Thuneberg L, (1982). Interstitial cells of Cajal: intestinal pacemaker cells. Adv Anat Embryol Cell Biol 71, 1-130 [Medline]

Tinel H, Cancela JM, Mogami H, Gerasimenko JV, Gerasimenko OV, Tepikin AV & Petersen OH (1999). Active mitochondria surrounding the pancreatic acinar granule region prevent spreading of inositol trisphosphate-evoked local cytosolic Ca²⁺ signals. EMBO J 18, 4999-5008 [Abstract/Full Text]

Torihashi S, Ward SM, Nishi K, Kobayashi S & Sanders KM (1995). c-kit-Dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract. Cell Tiss Res 280, 97-111

Vanderwinden JM & Rumessen JJ (1999). Interstitial cells of Cajal in human gut and gastrointestinal disease. Microsc Res Tech 47, 344-360 [Medline]

Ward SM, Burke EP & Sanders KM (1990). Use of rhodamine 123 to label and lesion interstitial cells of Cajal in canine colonic circular muscle. Anat Embryol 182, 215-224 [Medline]

Ward SM, Burns AJ, Torihashi S & Sanders KM (1994). Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine. J Physiol 480, 91-97 [Abstract]

Ward SMÖrdög T, Koh SD, Abu Baker S, Jun JY, Amberg G, Monaghan K & Sanders KM (2000). Pacemaking in interstitial cells of Cajal depends upon calcium handling by endoplasmic reticulum and mitochondria. J Physiol 525, 355-361 [Abstract/Full Text]

Ward SM & Sanders KM (1990). Pacemaker activity in septal structures of canine colonic circular muscle. Am J Physiol 259, G264-273 [Medline]

Ward SM & Sanders KM (1992). Dependence of electrical slow waves of canine colonic smooth muscle on calcium current. J Physiol 455, 307-319 [Abstract]

Acknowledgement

These experiments were supported by NIH DK 41315.

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Abe Y & Tomita T (1968). Cable properties of smooth muscle. J Physiol 196, 87-100	[Medline]
Burns AJ, Herbert TM, Ward SM & Sanders KM (1997). Interstitial cells of Cajal in the guinea-pig gastrointestinal tract as revealed by c-Kit immunohistochemistry. Cell Tiss Res 290, 11-20
Dickens EJ, Hirst GD & Tomita T (1999). Identification of rhythmically active cells in guinea-pig stomach. J Physiol 514, 515-531	[Abstract/Full Text]
Farkas DL, Wei M-D, Febbroriello P, Carson JH & Loew LM (1989). Simultaneous imaging of cell and mitochondrial membrane potential. Biophys J 56, 1053-1069	[Abstract]
Hirst GD, Bramich NJ, Teramoto N, Suzuki H & Edwards FR (2002). Regenerative component of slow waves in the guinea-pig gastric antrum involves a delayed increase in [Ca²⁺]_i and Cl^- channels. J Physiol 540, 907-919	[Abstract/Full Text]
Horiguchi K, Semple GSA, Sanders KM & Ward SM (2001). Distribution of pacemaker function through the tunica muscularis of the canine gastric antrum. J Physiol 537, 237-250	[Abstract/Full Text]
Horowitz BM, Ward SM & Sanders KM (1999). Cellular and molecular basis for electrical rhythmicity in gastrointestinal muscles. Ann Rev Physiol 61, 19-43	[Abstract/Full Text]
Huizinga JD, Farraway L & Den Hertog A (1991). Generation of slow-wave-type action potentials in canine colon smooth muscle involves a non-L-type Ca²⁺ conductance. J Physiol 442, 15-29	[Abstract]
Huizinga JD, Thuneberg L, Kluppel M, Malysz J, Mikkelsen HB & Bernstein A (1995). W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity. Nature 373, 347-349	[Medline]
Kim TW, Beckett EAH, Hanna R, Koh SDÖrdög T, Ward SM & Sanders KM (2002a). Regulation of pacemaker frequency in the murine gastric antrum. J Physiol 538, 145-157	[Abstract/Full Text]
Kim YC, Koh SD & Sanders KM (2002b). Voltage-dependent inward currents of interstitial cells of Cajal from murine colon and small intestine. J Physiol 541, 797-810	[Abstract/Full Text]
Koh SD, Jun JY, Kim TW & Sanders KM (2002). A Ca²⁺-inhibited non-selective cation conductance contributes to pacemaker currents in cultured interstitial cells of Cajal. J Physiol 540, 803-814	[Abstract/Full Text]
Koh SD, Sanders KM & Ward SM (1998). Spontaneous electrical rhythmicity in cultured interstitial cells of Cajal from the murine small intestine. J Physiol 513, 203-213	[Abstract/Full Text]
Langton P, Ward SM, Carl A, Norell MA & Sanders KM (1989). Spontaneous electrical activity of interstitial cells of Cajal isolated from canine proximal colon. Proc Natl Acad Sci U S A 86, 7280-7284	[Medline]
Lee HK & Sanders KM (1993). Comparison of ionic currents from interstitial cells and smooth muscle cells of canine colon. J Physiol 460, 135-152	[Abstract]
Nose K, Suzuki H & Kannan H (2000). Voltage dependency of the frequency of slow waves in antrum smooth muscle of the guinea-pig stomach. Jpn J Physiol 50, 625-633	[Medline]
Ördög T, Ward SM & Sanders KM (1999). Interstitial cells of Cajal generate electrical slow waves in the murine stomach. J Physiol 518, 257-269	[Abstract/Full Text]
Publicover NG & Sanders KM (1984). A technique to locate the pacemaker in smooth muscles. J Appl Physiol 57, 1586-1590	[Abstract]
Rumessen JJ & Thuneberg L (1996). Pacemaker cells in the gastrointestinal tract: interstitial cells of Cajal. Scand J Gastroenterol 31, 82-94
Sanders KM, (1996). A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract. Gastroenterology 111, 492-515	[Medline]
Sanders KMÖrdög T, Koh SD, Torihashi S & Ward SM (1999). Development and plasticity of interstitial cells of Cajal. Neurogastroenterol Motil 11, 311-338	[Medline]
Sanders KMÖrdög T, Koh SD & Ward SM (2000). A novel pacemaker mechanism drives gastrointestinal rhythmicity. News Physiol Sci 15, 291-298	[Abstract/Full Text]
Sanders KM, Stevens R, Burke E & Ward SM (1990). Slow waves actively propagate at submucosal surface of circular layer in canine colon. Am J Physiol 259, G258-263	[Medline]
Schinder AF, Olson EC, Spitzer NC & Montal M (1999). Mitochondrial dysfunction is a primary event in glutamate neurotoxicity. J Neurosci 16, 6125-6133	[Abstract/Full Text]
Smith TK, Reed JB & Sanders KM (1987). Origin and propagation of electrical slow waves in circular muscle of the canine proximal colon. Am J Physiol 252, C215-224	[Medline]
Suzuki H, Takano H, Yamamoto Y, Komuro T, Saito M, Kato K & Mikoshiba K (2000). Properties of gastric smooth muscles obtained from mice which lack inositol trisphosphate receptor. J Physiol 525, 105-111	[Abstract/Full Text]
Szurszewski JH, (1987). Electrical basis for gastrointestinal motility. In Physiology of the Gastrointestinal Tract, 2nd edn, ed. Johnson LR, pp. 383-422. Raven, New York
Thomsen L, Robinson TL, Lee JC, Farraway LA, Hughes MJ, Andrews DW & Huizinga JD (1998). Interstitial cells of Cajal generate a rhythmic pacemaker current. Nat Med 4, 848-851	[Medline]
Thuneberg L, (1982). Interstitial cells of Cajal: intestinal pacemaker cells. Adv Anat Embryol Cell Biol 71, 1-130	[Medline]
Tinel H, Cancela JM, Mogami H, Gerasimenko JV, Gerasimenko OV, Tepikin AV & Petersen OH (1999). Active mitochondria surrounding the pancreatic acinar granule region prevent spreading of inositol trisphosphate-evoked local cytosolic Ca²⁺ signals. EMBO J 18, 4999-5008	[Abstract/Full Text]
Torihashi S, Ward SM, Nishi K, Kobayashi S & Sanders KM (1995). c-kit-Dependent development of interstitial cells and electrical activity in the murine gastrointestinal tract. Cell Tiss Res 280, 97-111
Vanderwinden JM & Rumessen JJ (1999). Interstitial cells of Cajal in human gut and gastrointestinal disease. Microsc Res Tech 47, 344-360	[Medline]
Ward SM, Burke EP & Sanders KM (1990). Use of rhodamine 123 to label and lesion interstitial cells of Cajal in canine colonic circular muscle. Anat Embryol 182, 215-224	[Medline]
Ward SM, Burns AJ, Torihashi S & Sanders KM (1994). Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine. J Physiol 480, 91-97	[Abstract]
Ward SMÖrdög T, Koh SD, Abu Baker S, Jun JY, Amberg G, Monaghan K & Sanders KM (2000). Pacemaking in interstitial cells of Cajal depends upon calcium handling by endoplasmic reticulum and mitochondria. J Physiol 525, 355-361	[Abstract/Full Text]
Ward SM & Sanders KM (1990). Pacemaker activity in septal structures of canine colonic circular muscle. Am J Physiol 259, G264-273	[Medline]
Ward SM & Sanders KM (1992). Dependence of electrical slow waves of canine colonic smooth muscle on calcium current. J Physiol 455, 307-319	[Abstract]

				O. B. Balemba, A. C. Bartoo, M. T. Nelson, and G. M. Mawe Role of mitochondria in spontaneous rhythmic activity and intracellular calcium waves in the guinea pig gallbladder smooth muscle Am J Physiol Gastrointest Liver Physiol, February 1, 2008; 294(2): G467 - G476. [Abstract] [Full Text] [PDF]

				B. Lavoie, O. B. Balemba, M. T. Nelson, S. M. Ward, and G. M. Mawe Morphological and physiological evidence for interstitial cell of Cajal-like cells in the guinea pig gallbladder J. Physiol., March 1, 2007; 579(2): 487 - 501. [Abstract] [Full Text] [PDF]

				O. B. Balemba, M. J. Salter, T. J. Heppner, A. D. Bonev, M. T. Nelson, and G. M. Mawe Spontaneous electrical rhythmicity and the role of the sarcoplasmic reticulum in the excitability of guinea pig gallbladder smooth muscle cells Am J Physiol Gastrointest Liver Physiol, April 1, 2006; 290(4): G655 - G664. [Abstract] [Full Text] [PDF]

				G. Boddy and E. E. Daniel Role of L-Ca2+ channels in intestinal pacing in wild-type and W/WV mice Am J Physiol Gastrointest Liver Physiol, March 1, 2005; 288(3): G439 - G446. [Abstract] [Full Text] [PDF]

				S. M Ward, R. E. Dixon, A. de Faoite, and K. M Sanders Voltage-dependent calcium entry underlies propagation of slow waves in canine gastric antrum J. Physiol., December 15, 2004; 561(3): 793 - 810. [Abstract] [Full Text] [PDF]

				J. E. Bradley, U. A. Anderson, S. M. Woolsey, K. D. Thornbury, N. G. McHale, and M. A. Hollywood Characterization of T-type calcium current and its contribution to electrical activity in rabbit urethra Am J Physiol Cell Physiol, May 1, 2004; 286(5): C1078 - C1088. [Abstract] [Full Text] [PDF]

				G. Boddy, A. Bong, W. Cho, and E. E. Daniel ICC pacing mechanisms in intact mouse intestine differ from those in cultured or dissected intestine Am J Physiol Gastrointest Liver Physiol, April 1, 2004; 286(4): G653 - G662. [Abstract] [Full Text] [PDF]