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MUSCLE CELL BIOLOGY AND CELL MOTILITY

Spatial and temporal mapping of pacemaker activity in interstitial cells of Cajal in mouse ileum in situ

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

Submitted 6 September 2005 ; accepted in final form 19 December 2005

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Spontaneous electrical pacemaker activity occurs in tunica muscularis of the gastrointestinal tract and drives phasic contractions. Interstitial cells of Cajal (ICC) are the pacemaker cells that generate and propagate electrical slow waves. We used Ca²⁺ imaging to visualize spontaneous rhythmicity in ICC in the myenteric region (ICC-MY) of the murine small intestine. ICC-MY, verified by colabeling with Kit antibody, displayed regular Ca²⁺ transients that occurred after electrical slow waves. ICC-MY formed networks, and Ca²⁺ transient wave fronts propagated through the ICC-MY networks at 2 mm/s and activated attached longitudinal muscle fibers. Nicardipine blocked Ca²⁺ transients in LM but had no visible effect on the transients in ICC-MY. -Glycyrrhetinic acid reduced the coherence of propagation, causing single cells to pace independently. Thus, virtually all ICC-MYs are spontaneously active, but normal activity is organized into propagating wave fronts. Inhibitors of dihydropyridine-resistant Ca²⁺ entry (Ni²⁺ and mibefradil) and elevated external K⁺ reduced the coherence and velocity of propagation, eventually blocking all activity. The mitochondrial uncouplers, FCCP, and antimycin and the inositol 1,4,5-trisphosphate receptor-inhibitory drug, 2-aminoethoxydiphenyl borate, abolished rhythmic Ca²⁺ transients in ICC-MY. These data show that global Ca²⁺ transients in ICC-MYs are a reporter of electrical slow waves in gastrointestinal muscles. Imaging of ICC networks provides a unique multicellular view of pacemaker activity. The activity of ICC-MY is driven by intracellular Ca²⁺ handling mechanisms and entrained by voltage-dependent Ca²⁺ entry and coupling of cells via gap junctions.

Ca²⁺ signaling; slow waves; gastrointestinal motility

In the small intestine, pacemaker activity arises from the area between the circular and longitudinal muscle (LM) layers [myenteric (MY) region; see Ref. 31], and ICCs form a dense network in this region (ICC-MY; 39). ICC-MYs are electrically coupled to each other and to neighboring smooth muscle cells (3). Pacemaker activity is generated and actively spreads within the ICC network and conducts passively to the surrounding smooth muscle syncytium (13). The mechanism required for generation and propagation of slow waves within ICC networks has been the subject of considerable debate (see Ref. 25). There is broad agreement that pacemaker currents are initiated via release of Ca²⁺ from inositol 1,4,5-trisphosphate (IP₃) receptor-operated stores; however, the nature and Ca²⁺ dependence of the ionic conductance responsible for pacemaker current is controversial. Some investigators have suggested that a Ca²⁺-activated Cl^– conductance is responsible for pacemaker current (11, 17), whereas others have suggested that nonselective cation conductances are responsible (8, 19, 41). Evidence suggesting the involvement of Ca²⁺-activated Cl^– currents has come largely from studies using Cl^– channel-blocking drugs that also block nonselective cation channels (see Ref. 19). However, voltage-clamp studies of ICC, where the nature of ionic conductances in ICC can be determined, have also been criticized because much of this work has been performed on cultured networks of ICC, in which proper space clamp is questionable, and there is always the possibility of phenotypic changes in culture.

Another feature of pacemaker activity that has been controversial is the mechanism by which slow waves actively propagate (or entrain the activity of other ICC) in ICC-MY networks. We have observed dihydropyridine-resistant, voltage-dependent Ca²⁺ currents in fresh (21) and cultured (16) ICC and proposed that voltage-dependent Ca²⁺ entry might trigger (or phase advance) pacemaker events in coupled ICC. Others have suggested that depolarization initiates IP₃ synthesis or increases coupling between G protein-coupled receptors and IP₃ receptors (3, 7, 15, 38). However, neither of these latter two phenomena has been shown to occur in ICC.

In the present study, we have sought to study pacemaker activity in ICC networks of the murine small intestine in situ using Ca²⁺ fluorescence imaging (9, 10, 30, 33). We have explored the mechanism of pacemaker generation, coupling between ICC, and propagation. Our data show that fluorescence transients using the indicator fluo-4 are a good reporter of electrical slow wave activity. The use of this technique to investigate pacemaker activity in networks provides a unique macroscopic view of the interactions between coupled ICC and cell-to-cell propagation.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Animals and tissue preparation. BALB/c, W/W^V-mutant mice, and wild-type male and female siblings (WBB6F1/J-Kit⁺/Kit⁺) between the ages of 30 and 60 days (Jackson Laboratory, Bar Harbor, ME) were used. The mice were euthanized by administering isoflurane inhalation (Baxter, Deerfield, IL), followed by cervical dislocation. The use and treatment of animals was approved by the Institutional Animal Use and Care Committee at the University of Nevada.

A midline abdominal incision was made, and a segment of terminal ileum (3–4 cm in length) was removed and opened along the mesenteric border. The luminal contents were flushed away with Krebs-Ringer buffer (KRB) solution, and the preparation was pinned with the mucosa facing down in a Sylgard-lined organ bath. Strips of LM and serosa were peeled away with the use of fine forceps in the center region of the preparation to reveal the underlying ICC-MY network that remained attached to the circular muscle (CM). For simultaneous recordings of Ca²⁺-induced fluorescence and membrane potential during slow wave activity, the mucosa and submucosa were peeled off from the CM.

The organ bath was maintained at 36.0 ± 0.5°C and was continuously perfused with warmed, oxygenated KRB solution. The tissue was left to equilibrate for at least 1 h before the dye-loading procedure (see below), during which time rhythmic contractions became evident.

Solutions and drugs. The KRB solution contained (in mM) 120.4 NaCl, 5.9 KCl, 15.5 NaHCO₃, 11.5 glucose, 1.2 MgCl₂, 1.2 NaH₂PO₄, and 2.5 CaCl₂. This solution had a pH of 7.3–7.4 at 37°C when bubbled with 97% O₂-3% CO₂. DMSO, cremophor EL, nifedipine, nicardipine, atropine, Ni²⁺, cyclopiazonic acid (CPA), TTX, pinacidil, carbonyl cyanide FCCP, ryanodine, -glycyrrhetinic acid (-GA), and 2-aminoethoxydiphenyl borate (2-APB) were obtained from Sigma-Aldrich (St. Louis, MO). Mibefradil dihydrochloride was a gift from Dr. Eva-Maria Gutknecht and Dr. Pierre Weber (Hoffman-LaRoche, Basel, Switzerland). 2-APB, CPA, FCCP, pinacidil, and ryanodine were dissolved in 0.1% DMSO. All other drugs were dissolved in water.

Fluorescent dye loading. After equilibration, the tissues were incubated with 25 µg of fluo-4 (FluoroPure AM; Molecular Probes, Eugene, OR) in a solution of 0.02% DMSO and 0.01% nontoxic detergent Cremophor EL for 20 min at 2.5°C. The dye primarily loaded into the ICC-MY network and any remaining LM fibers. After incubation, the tissues were perfused with warmed Krebs solution (36.0 ± 0.5°C) for 20 min to allow for deesterification and trapping of the dye in cells. Fields of view were selected that contained well-loaded ICC-MY and that displayed Ca²⁺ oscillations. Because the fields of view often contained out-of-focus regions or areas that appeared to be poorly loaded, these areas were not included in the analysis. For most experiments, control activity was recorded (40 s), a stimulus was delivered for 10 min during a period of nonillumination, and then activity was recorded again for 40 s. In experiments using TTX, activity was recorded after 5 min.

Equipment. Preparations were viewed under a microscope (model BX50WI; Olympus, Melville, NY) fitted with an epifluorescence device. Light was supplied by a 100-W high-pressure mercury burner. Water-immersion lenses (x20, x40; Olympus UMPlanF) were also used. Neutral density filters were used to adjust excitation and emission light intensities. A wide-interference blue filter cube (U-MWIB) produced excitation between 460–490 nm, and emission >515 nm, suitable for fluo-4 (peaks: excitation 490 nm, emission 515 nm). Ca²⁺-induced fluorescence was recorded using a video-rate iCCD camera (30 frames/s; IC-300B; Photon Technology International, Monmouth Junction, NJ) and a digital video recorder (DHR-1000; Sony) or a Cascade 512B camera (15.6 frames/s; Roper Scientific, Trenton, NJ), and MetaMorph 6.26 software (Universal Imaging/Molecular Devices, Downingtown, PA).

For simultaneous recording of Ca²⁺-induced fluorescence and voltage, an inverted Eclipse TS100F microscope (Nikon, Melville, NY), Lambda LS (Sutter Instrument, Novato, CA) light source, and a Nikon Fluor lens (x20) was used. Impalements were made with the use of sharp intracellular electrodes, and the membrane potential was recorded using a Digidata 1322A Axoprobe-1A amplifier, AxoScope version 0.2.05 (Axon Instruments/Molecular Devices, Union City, CA) and Imaging Workbench 5.1.28 software (INDEC BioSystems, Mountain View, CA). Voltage traces were sampled at 1 kHz, and Ca²⁺ traces were sampled at 18.2 frames/s. Ca²⁺ traces were smoothed (±55 ms), the pair of points straddling the 33% level were located, and the slope was calculated. The exact time at which 33% of maximum amplitude was reached was interpolated from the slope.

We purposely picked ICC-MY for analysis of Ca²⁺ transients that were in parallel to both the site of impalement and the wave front of propagation. Cells along a wave front were activated in a nearly simultaneous manner. Thus a small distance between points along a wave front would produce minimal errors in latency. However, in the worst case, the maximum distance separating electrodes and ICC-MY, from which Ca²⁺ transients were tabulated was 20 µm (usually 5–10 µm). With the propagation velocities measured in ICC-MY networks (see RESULTS), the maximum error associated with the spatial distance between sites of recording was a fraction of the total delay between the upstrokes of slow waves and Ca²⁺ transients.

Image processing and analysis. If movies were stored on a digital videotape, small clips (40–60 s) were captured using a digital video (DV:IEEE-1394) interface to a Macintosh G4 or G5 computer (Apple, Cupertino, CA). Movies saved directly to disk required no conversion. Image sequences were converted to 8-bit gray scale, calibrated, and analyzed using custom-built software (Volumetry 1.2; GWH).

Because we did not limit the movement of the tissue during the experimental procedure, there was often movement associated with Ca²⁺ waves. Tracking routines were used to motion stabilize each cell in the field of view from which Ca²⁺ measurements were made.

Several imaging techniques were used to better visualize Ca²⁺ transients and are displayed in the bottom left corner images of figures and include the following: 1) Z_Frame, an individual frame taken from an image sequence; 2) Z_dIdT, differentiated frame (t specified) from an image sequence; 3) Z_avg, the average of all images in an image sequence (z-axis); 4) Z_STDEV, the SD of fluorescence intensity in an image sequence; 5) X_avg, spatiotemporal map of fluorescence intensity by averaging along rows of pixels (x-axis); and 6) Y_avg, spatiotemporal map of fluorescence intensity by average down columns of pixels (y-axis).

The fluorescence intensity was measured using the fluorescence ratio (F/F_avg), which compares Ca²⁺ activity in a cell with its average fluorescence calculated from the entire image sequence, instead of with an arbitrary resting fluorescence reference (F₀). This minimizes offset and bleaching errors and is appropriate for a continuously oscillating signal (10). The frequency (interval), amplitude, duration, rising slope, and decay constant of Ca²⁺ transients were measured. To calculate the velocity of propagation, the distance and angle of cell pairs were calculated. Pairs of cells in parallel to the direction of propagation had the slowest velocity. The sequence of activation and stability of propagating pacemaker activity wave front were also calculated (see Fig. 3).

View larger version (64K): [in this window] [in a new window]   Fig. 3. A: active ICC-MY cell bodies and processes and longitudinal muscle (LM) fibers (horizontal dark bands) in a field of view. Ca2+-induced fluorescence in selected cells (see colored circles) was measured. B: the progression of activity through the ICC-MY network, and activation of LM (white horizontal bands) with the direction of propagation denoted by the direction of the arrow in the first frame. ZFrame, individual frame taken from an image sequence. C: Ca2+ transients from selected cells. There was a brief period in which a small region of the network (left-hand edge) fired prematurely and pacemaker activity did not propagate across the field of view (***). D: the time at which cells initiated Ca2+ transients as the wave front propagated across the field of view. E: the sequence of activation for the selected cells. F: velocity between each pair of cells (nonrepeating, positive velocities: y-axis) and the angular position of the cells in relation to one another (angle: x-axis) was calculated and plotted. The direction of propagation of the pacemaker activity was determined by finding the angle at which the minimum velocity was recorded (see arrow). This velocity equates to the speed of propagation (perpendicular to the wave front) because cells aligned in parallel to the wave front were activated nearly simultaneously, resulting in high velocities. G: time delay between cells with the length of the colored bar corresponding to the time delay from the activation of the previous cell in the sequence (see arrows corresponding to activation of orange cell, followed by activation of yellow cell in wave 3; see D). While the speed of propagation across the field of view varied considerably (overall length of bars in G), the sequence of activation remained similar.

Verification of ICC using kit labeling. After Ca²⁺ fluorescence was recorded from an active region of ICC-MY network, nifedipine (1 µM) was added to minimize contractile activity and movement. ACK2, an antibody that recognizes extracellular domain of the protein kit, was preconjugated to a fluorescence label using the 594 Tagging Kit (Molecular Probes) and was added to the preparation every 10 min for 30 min (3 µl/1 ml Krebs solution, 100 µl; see Ref. 39). The preparation was then washed for 15 min with Krebs solution containing nifedipine (1 µM). Labeled ICC were visualized (peaks: excitation 560 nm, emission 645 nm; Filter Set 41004, Chroma Technology; Rockingham, VT) and compared with the positions of cells that generated spontaneous Ca²⁺-dependent fluorescence signals (see Fig. 1).

View larger version (76K): [in this window] [in a new window]   Fig. 1. A: Ca2+-induced fluorescence averaged >0.5 s during the upstroke of a Ca2+ transient in a cluster of cells. These cells were Kit positive (B: white arrowheads) and can be considered interstitial cells of Cajal in the myenteric region (ICC-MY). To better reveal only those cells that displayed Ca2+ transients, the standard deviation of fluorescence intensity throughout the image sequence was calculated (C: dark gray = greater deviation of intensity). The perinuclear regions of ICC-MY displayed more intense, longer-lasting Ca2+ transients than in cell processes (D and E). Spatio-temporal maps were used to summarize fluorescence activity in a field of view and show rhythmic Ca2+ waves (vertically oriented white streaks) associated with small distortions of the tissue (F: white arrow). After 10 min, there was a decrease in the amplitude of Ca2+ transients in individual cells, but frequency was not significantly altered (G).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Spontaneous Ca²⁺ transients in ICC-MY. Image sequences of Ca²⁺-induced fluorescence in branched cells within the myenteric region of the murine small intestine were recorded and displayed rhythmic Ca²⁺ transients at an average frequency of 27.17 ± 1.04 cpm (0.45 ± 0.017 Hz; interval 2.21 ± 0.08 s, n = 25, cells = 100; Fig. 1). The activities of individual cells could be visualized, and the average variation in frequency of spontaneous Ca²⁺ transients in each cell was 12.5%. The frequency of Ca²⁺ transients was similar to the frequency of electrical pacemaker activity in the murine ileum (36, 39). For most fields of view, wave fronts of Ca²⁺ transients spread through networks consisting of many cells. Of course, it was not possible to follow this activity uniformly through fields of view that contained regions that were out of focus or were poorly loaded with the Ca²⁺ indicator.

During the Ca²⁺ transients, the morphology of fluo-4-loaded cells in the myenteric region was apparent. These cells had oval or triangular cell bodies and several fine processes that contacted neighboring cells to form a network. The Ca²⁺ transients spread through the network. The cells with rhythmic Ca²⁺ transients had morphologies and anatomic positions identical to ICC-MY (34, 36, 39). However, we also performed double-labeling experiments with fluorescently tagged Kit antibody to ensure that the cells displaying rhythmic Ca²⁺ transients were ICC-MY. In these experiments, nicardipine (1 µM) was added to reduce contractions and movement. Nicardipine alone did not significantly affect the rhythmic Ca²⁺ transients. After imaging Ca²⁺ transients in a given region, tissues were incubated with Kit antibody (see MATERIALS AND METHODS) and revisualized. Cells in the myenteric region with rhythmic Ca²⁺ transients were Kit positive, confirming the cells were ICC-MY (Fig. 1, A and B). We also visualized Ca²⁺ transients in Kit-positive cells from small intestinal muscles of wild-type (WBB6F1/J-Kit⁺/Kit⁺) mice and looked for similar events in the same region of tissues of W/W^V mice that lacked ICC-MY (e.g., Ref. 39). Branched, multiprocessed cells with spontaneous Ca²⁺ transients were not observed in the myenteric region of the small intestines of W/W^V mice (not shown).

Because several of our studies tested the effects of drugs or other chemicals on the pacemaker activity in the small intestine, we initially performed time controls with vehicles (e.g., 0.1% DMSO) to characterize the bleaching of Ca²⁺ fluorescence under the conditions of our experiments. We observed a consistent decline in the amplitudes of Ca²⁺ transients recorded from ICC-MY that averaged 37.7 ± 3.3% (n = 5, cells = 15) during 10 min between exposures to the excitation light. There was no significant change in frequency of Ca²⁺ transients during the same time period (i.e., initial interval averaged 2.80 ± 0.13 s compared with an interval of 2.53 ± 0.15 s, P > 0.05; after 10 min; Fig. 1, F and G).

Ca²⁺ transients in ICC-MY were associated with electrical slow waves. Because the frequency of rhythmic Ca²⁺ transients in ICC-MY was similar to the frequency of electrical slow waves, we made simultaneous intracellular recordings of electrical activity while imaging from the same region of tissue to determine whether these events were related. Nicardipine (1 µM) was added in these experiments to reduce Ca²⁺-induced fluorescence in CM and limit movement of the preparations. Impalements of CM cells within 5–20 µm of ICC-MY displaying rhythmic Ca²⁺ transients demonstrated a 1:1 relationship between slow waves and Ca²⁺ transients. The upstrokes of slow waves occurred first, and Ca²⁺ transients followed with an average delay of 58 ± 11 ms (33% amplitude, n = 6, cells = 30; Fig. 2). These data suggest that Ca²⁺ transients, when resolvable, are a reliable reporter of slow wave activity, and slow waves appear to initiate the rise in Ca²⁺ in ICC-MY.

View larger version (31K): [in this window] [in a new window]   Fig. 2. A: trace of membrane potential (black) recorded from circular muscle (CM) and Ca2+-induced fluorescence (orange trace) in a small region of the ICC-MY network during slow wave activity. B: nicardipine (1 µM) was present to block Ca2+-induced fluorescence generated by slow waves in the CM. C: depolarization was detected (50 ms) before a subsequent rise in Ca2+-induced fluorescence.

TTX (1 µM, n = 5, cells = 25) or atropine (1 µM, n = 5, cells = 25) had no effect on the frequency of spontaneous Ca²⁺ transients in ICC-MY networks (P > 0.1). TTX, however, appeared to improve the regularity (i.e., change in interval event to event) of Ca²⁺ transients from 0.29 ± 0.05 s to 0.14 ± 0.03 s (P = 0.04).

Pacemaker activity wave front. A wave front of pacemaker activity could be reconstructed by locating Ca²⁺ transients in ICC-MY that occurred nearly simultaneously within the field of view. The direction of spread of pacemaker activity was perpendicular to the wave front. For each experiment, the direction of propagation differed because pacing was spontaneous. The average speed of propagation in control experiments was 1.9 ± 0.1 mm/s (n = 12). We had sufficient temporal and spatial resolution to tabulate the sequence of activation of individual ICC-MY as wave fronts propagated. The sequence and direction (i.e., change of 5.4 ± 0.9° from wave to wave) of propagation were relatively consistent during the 40-s recordings in contrast to propagation in gastric ICC-MY networks; see Ref. 9); however, the delays between activation of cells in the sequence varied considerably, altering the overall velocity by up to 200% (average change in velocity per wave = 30%). This suggests that when a stable pacing site emerges, the propagation pathway through a network of ICC is relatively stable; however, the excitability of individual ICC-MY regulates the time at which individual cells become active (Fig. 3).

Coupling between ICC-MY and LM. In most preparations, LM fibers were peeled away to allow direct visualization of the ICC-MY network. In some cases, a few LM fibers or small bundles remained after dissection, and it was possible to measure Ca²⁺ transients from both cell types and determine the sequence of activation. LM cells were activated after ICC-MY with an apparent delay averaging 126 ± 13 ms (n = 6, 72 transients; Fig. 4). It should be noted that it is difficult to calculate a precise delay between ICC-MY and LM activation because it is impossible to know the exact site at which the LM comes to threshold. The delay was not constant and varied considerably from event to event. In one experiment (3% of analyzed transients), the Ca²⁺ transient in a bundle of LM appeared to precede activation of adjacent ICC-MY. It is likely that the LM cells in this case were activated from ICC-MY outside the field of view. The sequence of activation (i.e., ICC-MY to LM) is consistent with the results of simultaneous electrical measurements from ICC-MY and adjacent CM or LM cells in the small intestine (17) and stomach (11a), suggesting that ICC-MY drive the activity of smooth muscle cells.

View larger version (53K): [in this window] [in a new window]   Fig. 4. A: averaged Ca2+-induced fluorescence in a field of view in which elongated LM (horizontal) and CM (vertical) fibers are visible with ICC-MY cells. Differentiating the image sequence to reveal rapid changes in fluorescence (B) demonstrated that the ICC-MY network became active 150 ms before the muscle fibers (C) fired. This is portrayed in a spatiotemporal map of Ca2+-induced fluorescence taken from the yellow rectangle (STMap: D), in which a faint band of fluorescence (ICC-MY arrow) precedes the intense band of fluorescence produced by muscle fibers becoming active (LM arrow). E and F: traces show Ca2+-induced fluorescence intensity taken from ICC-MY (blue) and LM (orange). The initial rise in Ca2+-induced fluorescence in LM (* in F) is an artifact due to underlying fluorescence in the ICC-MY network that could not be excluded from the region of interest. The projected activation time of the LM is denoted by the dashed line. ZdIdT, differentiated frame (t specified) from an image sequence.

-GA reduces coherence of pacemaker propagation in networks of ICC-MY.

View larger version (63K): [in this window] [in a new window]   Fig. 5. A: active ICC-MY in a field of view from which control activity was recorded. B: the field of view in which activity after -glycyrrhetinic acid (10 µM) was recorded. C: during control activity (STMap), ongoing, repetitive waves of pacemaker activity were visualized as vertically orientated white streaks. D: after the addition of -glycyrrhetinic acid (STMap, 10 µM), no coherent waves of pacemaker activity were observed and cells displayed Ca2+ transients temporally unrelated to neighboring cells. Traces taken from individual ICC-MY (see colored circles) during control activity (E) and after -glycyrrhetinic acid (F) show the transformation to irregular, uncoordinated Ca2+ transients but little reduction in the amplitude of transients.

Ni²⁺ (30 µM) reduced the frequency (n = 5, cells = 20; interval 167 ± 20%, amplitude 57 ± 7%) and severely disrupted Ca²⁺ transients at 100 µM (n = 3, cells = 10; interval 244 ± 10%, amplitude 56 ± 6%). In control conditions, pacemaker activity propagated through all cells in networks, but after Ni²⁺, the coherence of propagation was greatly reduced and propagation was restricted to small clusters of cells. Ni²⁺ (30 µM) decreased the average rate of rise of Ca²⁺ transient by 59.9 ± 8.5% (n = 5), and in networks with remaining propagated events, the velocity decreased from 1.6 ± 0.06 to 0.6 ± 0.03 mm/s (n = 3) (Fig. 6).

View larger version (53K): [in this window] [in a new window]   Fig. 6. A: active ICC-MY in a field of view from which Ca2+ transients were extracted (colored circles). Control activity (B and D) shows ongoing waves of pacemaker activity. Ca2+ transients exhibited a rapid upstroke phase followed by a more variable plateau phase (D). After the addition of Ni2+ (100 µM), the frequency of rhythmic activity was decreased and cells failed to generate a rapid upstroke (C and E). With time, the amplitude of Ca2+ transients decreased to the point where they were not possible to resolve. Before total loss of Ca2+ transients in the presence of Ni2+, Ca2+ transients could be observed in cell bodies of ICC-MY but were too faint to be resolved in processes (F). Note: the rapid upstroke phase of the Ca2+ transients was significantly reduced by Ni2+ (D compared with E).

In separate experiments where the effect of mibefradil was monitored as the drug was added to the perfusing solution and the final concentration was increased slowly, mibefradil reduced coherence of pacemaker propagation in ICC-MY networks, and Ca²⁺ transients became slow, irregular, and did not spread through all of the ICC-MY within a given field (Fig. 7, A–C). These results indicate that a voltage-dependent Ca²⁺ conductance, primarily due to a dihydropyridine-resistant conductance in ICC-MY, is critical for propagation of pacemaker activity within networks of ICC.

View larger version (38K): [in this window] [in a new window]   Fig. 7. A: continuous recording from a representative ICC-MY to which mibefradil (0.1 µM) was added. After 20 s, regular ongoing Ca2+ transients were reduced in amplitude and the interval was increased. Typically, at concentrations of 0.1–1 µM mibefradil, all activity was blocked after 10 min. B and C: Ca2+ transients from several cells before (B) and after application of 0.1 µM mibefradil. At lower concentrations of mibefradil (0.05 µM), Ca2+ transients were still observed after 10 min but at a decreased frequency [control (D) compared with 0.05 µM mibefradil (E)]. To minimize bleaching, experiments were performed in which 0.05 µM mibefradil was added without a control (F) and then nicardipine (1 µM) was added (G). Nicardipine further increased the interval between Ca2+ transients, often blocking activity altogether.

Effect of pinacidil and high K⁺. The effects of altering membrane potential on entrainment of Ca²⁺ transients was tested using a K⁺ channel agonist and by elevating external K⁺. Pinacidil, which activates ATP-sensitive K⁺ channels, hyperpolarizes ICC-MY of the murine small intestine (18). Pinacidil (10 µM) did not block or reduce the overall frequency of Ca²⁺ transients in the ICC-MY (interval 102 ± 15%, amplitude was reduced by 50 ± 3%; n = 5); however, spatiotemporal maps of ICC-MY networks showed that in some cases, the activity did not propagate throughout the network coherently (Fig. 8, D and E). Pinacidil blocked all Ca²⁺ transients in the LM layer (not shown). Velocities of propagation were not statistically different from controls (control = 2.7 ± 0.4 mm/s compared with pinacidil, 2.2 ± 0.3 mm/s; n = 3).

View larger version (56K): [in this window] [in a new window]   Fig. 8. A: active ICC-MY in a field of view to which pinacidil (10 µM) was later added. B: active cells in a field of view to which K+ (12 mM) was later added. Pinacidil (10 µM) did not significantly alter the frequency of Ca2+ transients compared with controls (control: C compared with pinacidil: D). This example shows a period in which the orderly propagation of pacemaker activity through the ICC-MY network was briefly disrupted due to the appearance of separate pacing sites (see dysrhythmic events in D denoted by *** beneath the spatiotemporal map; these events also appear as fluorescence traces in E). Similar disruptions were occasionally observed in controls. F: individual frames during propagation of pacemaker activity through the field of view in the presence of pinacidil (10 µM). G: control activity in ICC-MY network with 2 prominent bands of LM, giving rise to the two intermittent bright bands in the STMap. After the addition of high K+ (12 mM), both the interval and amplitude of Ca2+ transients in ICC-MY became irregular (H and I). J: individual frames during a wave of pacemaker activity after the addition of K+ (12 mM).

Effects of disrupting intracellular Ca²⁺ handling. Slow wave generation in ICC involves regulation of intracellular Ca²⁺ in a compartment near the plasma membrane ("pacemaker unit"; see Ref. 26). Ca²⁺ handling has been suggested to occur via IP₃ receptor-operated Ca²⁺ release from internal stores and Ca²⁺ uptake into mitochondria (41). These conclusions came largely from studies of cultured ICC, so we tested the effects of several drugs known to interfere with intracellular Ca²⁺ handling on rhythmic Ca²⁺ transients in ICC-MY networks in situ.

Ryanodine (10 µM: n = 4) had no significant effect on the frequency of Ca²⁺ transients (interval 94 ± 3%, amplitude reduced by 44.7 ± 2%). 2-APB (n = 5) (50 µM), significantly slowed the frequency of Ca²⁺ transients in ICC-MY (interval: 544 ± 131%, P < 0.05); however, when a transient was observed (in 3 of 5 tissues) the amplitudes were enhanced (amplitude increased to 160 ± 41%; Fig. 9). Evidence for the involvement of Ca²⁺ stores in the generation of Ca²⁺ transients was also obtained by testing CPA (10 µM, n = 5), an inhibitor of sarcoplasmic reticulum Ca²⁺-ATPase pump. This compound slowed the frequency of transients (interval: 201 ± 74%) and reduced the amplitude (by 25 ± 14%) of Ca²⁺ transients in ICC-MY (data not shown). The mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP, 1 µM, n = 4), and antimycin A (10 µM; n = 3; not shown), an inhibitor of complex 3 in the mitochondrial electron transport chain, abolished Ca²⁺ transients (Fig. 10).

View larger version (49K): [in this window] [in a new window]   Fig. 9. A: active ICC-MY in a field of view to which ryanodine (10 µM) was later added. B: active ICC-MY in a field of view in another experiment to which 2-aminoethoxydiphenyl borate (2-APB; 50 µM) was later added. There was no significant change in frequency of Ca2+ transients in ICC-MY after ryanodine treatment (control: C compared with D and E: ryanodine). F: the progression of pacemaker activity through the network in the presence of ryanodine (10 µM) as individual frames from the image sequence. Ca2+ transients in ICC-MY after the addition of 2-APB (50 µM) were either blocked or occurred infrequently with larger amplitudes (control: G compared with H and I: 2-APB). J: the progression of activity shown in individual frames after 2-APB addition (50 µM).

View larger version (67K): [in this window] [in a new window]   Fig. 10. A: active ICC-MY to which FCCP (1 µM) was later added. B: individual frames of the progression of a pacemaker wave in a control recording. After the addition of FCCP (1 µM), all activity was blocked (control: C compared with D and E: FCCP).

	DISCUSSION

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A previous study used imaging techniques to study the activity in murine small intestinal muscles treated with wortmannin and cytochalasin D to reduce contractile activity (43). These authors observed rhythmic Ca²⁺ oscillations in ICC at 9 per min. At the temperatures used in their studies (29–31°C) the frequency of Ca²⁺ oscillations was far below the slow wave frequency in the murine ileum at 30°C (i.e., 22/min; Ward SM, unpublished observations). Yamazawa and Iino (43) also reported a relatively poor correlation (8 of 21 muscles) between Ca²⁺ transients in ICC-MY and LM. The very slow frequencies of Ca²⁺ transients and the poor correlation with smooth muscle events suggest that the activity recorded previously may not have been related to electrical slow waves. In the present study, we made simultaneous measurements of spontaneous Ca²⁺ transients in ICC-MY and electrical slow wave activity and showed that these events were coupled in a 1:1 manner. The initial upstroke of slow wave potentials was followed, after a short latency, by Ca²⁺ transients in ICC-MY. An important corollary to this observation is that global Ca²⁺ transients in ICC-MY are a response to depolarization rather than the signal that initiates slow waves. Our data demonstrate that global intracellular [Ca²⁺] can be an effective reporter of slow wave activity in GI muscles and that measuring Ca²⁺ transients with video microscopy provides a unique multicellular view of pacemaker activity in networks of ICC and in ICC-to-smooth muscle coupling. Caution must be applied to data in which global Ca²⁺ transients are blocked, however, because it may be possible to reduce the global Ca²⁺ transients to an unresolvable level before electrical slow waves are completely inhibited.

Many features of the Ca²⁺ transients in intact ICC-MY networks are consistent with the properties of slow waves in intact GI muscles and organs. For example, we found that the Ca²⁺ transients in ICC-MY were spontaneous and not dependent upon neural inputs. The propagation rate of Ca²⁺ transients in networks of ICC was not "nearly synchronous," as recently reported in studies of Ca²⁺ transients in ICC of the guinea pig pylorus (37). In fact, propagation was relatively slow compared with propagation of electrical activity in many excitable tissues, and rates were similar to slow wave propagation rates in GI muscles (28; 4–35 mm/s). Active propagation in ICC-MY networks and conduction of slow waves to smooth muscle cells appears to depend on coupling between ICC-MY and coupling between ICC-MY and smooth muscle cells. Propagation also depends upon voltage-dependent, dihydropyridine-resistant Ca²⁺ entry. Imaging of many cell networks has important advantages over electrophysiological studies, because this approach can provide insights into the sequence of cell-to-cell activation, the means of communication between ICC, the means of communication between ICC and smooth muscle cells, and the regulatory effects of neurotransmitters, hormones, and paracrine substances on pacemaker activity.

The sequence of events (i.e., electrical slow waves preceded Ca²⁺ transients) suggests that global Ca²⁺ transients are a response to slow wave depolarization rather than an initiator signal that generates pacemaker activity. ICCs express voltage-dependent Ca²⁺ channels (16, 21) that would result in Ca²⁺ influx in response to activation by depolarization. Our data showing that Ni²⁺ and mibefradil block Ca²⁺ transients suggest that Ca²⁺ entry is responsible for the global Ca²⁺ transients we observed. Previous studies have suggested that slow waves are initiated by highly localized changes in intracellular [Ca²⁺] due to release of Ca²⁺ from IP₃ receptor-operated stores (e.g., Refs. 25, 41). Our evidence from previous studies (19, 41) suggests that release of Ca²⁺ from IP₃ receptor-operated stores triggers Ca²⁺ uptake into mitochondria, a transient and highly localized depression of Ca²⁺ in pacemaker units, and activation of nonselective cation channels. Depolarizations (called "unitary potentials"; see Ref. 11a) from this inward current summate, and this signal initiates voltage-dependent Ca²⁺ entry through dihydropyridine-sensitive Ca²⁺ channels and the global Ca²⁺ signals we have observed in the present study. The localized Ca²⁺ transients from IP₃ receptors that initiate spontaneous activity, if not amplified by depolarization and Ca²⁺ entry, may be difficult to resolve in ICC-MY.

We have not been able to image ICC-MY and obtain simultaneous intracellular electrical recordings from the same cells being imaged. Therefore, to test the temporal sequence between Ca²⁺ transients and electrical slow waves, we settled for impalement of CM cells that were within 5–20 µm of ICC-MY with rhythmic Ca²⁺ transients. Previous studies (3, 6, 17) have shown that slow waves occur in ICC-MY before the events can be recorded in adjacent smooth muscle cells, making the case that slow waves originate in ICC-MY. It should be noted that the spatial separation between cells imaged and the cells from which electrical recordings were made would have introduced errors in the latencies measured between the slow waves and Ca²⁺ transients. Conduction of slow waves from ICC-MY to smooth muscle cells is electrotonic (3), so the delay between initiation of a slow wave in an ICC and an adjacent smooth muscle cell is small, but the resistance and capacitance properties of the ICC and smooth muscle syncytia increase the latency as a function of distance between the sites of recording. Our results show that slow wave events, initiated in the ICC-MY network, conduct to the smooth muscle cells before Ca²⁺ transients develop in the ICC. The delay between initiation of a slow wave in the ICC network and the voltage response in the impaled smooth muscle cell is not included in the latencies we measured between slow waves and Ca²⁺ transients. Thus the time between the upstrokes of slow waves and the development of Ca²⁺ transients in ICC-MY is underestimated by our measurements.

ICC express connexin genes and proteins, and gap junctions between ICC can be observed with electron microscopy (3, 4, 12, 29). Dye coupling also occurs within ICC networks (see Refs. 6 and 17). Although it has been commonly assumed that gap junctions provide coupling within ICC networks, this has not been demonstrated directly. In this study, we found that -GA, a well-known gap junction uncoupler, disrupted coherence of pacemaker propagation, and blocked cell-to-cell propagation of Ca²⁺ transients. These studies also demonstrate, for the first time, that essentially all of the cells in an ICC-MY network in situ have intrinsic pacemaker activity. When the cells are uncoupled, they generated Ca²⁺ transients spontaneously. The data show that "entrainment" or coordination of the intrinsic pacemaker activities of the multitude of ICC that form a network produces the phenomenon of active propagation.

The mechanism of slow wave entrainment has been an object of debate for many years. Some investigators (37) have suggested that slow wave activity is best described by an array of coupled oscillators, and this concept has recently been revived as a model for rhythmicity in small strips of pyloric smooth muscle. We have argued in previous studies (24) that coupled oscillator models, as presented, do not adequately simulate the important properties of electrical slow waves or provide discrete variables representing real-life parameters (such as membrane resistance and capacitance, ionic permeabilities, nonlinear changes in membrane conductance, and junctional resistance) that can be manipulated to simulate biological experiments. In the present study, we have provided support for the hypothesis that coherent propagation of pacemaker activity depends on voltage-dependent mechanisms. Propagation of activity is constrained by cable properties of the ICC network, such as cell-to-cell coupling and input resistance, and the propagation mechanism works in conjunction with the cellular oscillator mechanism. On the basis of studies of cultured ICC from the small intestine, we suggested that pacemaker activity in ICC depends on closely associated cellular structures, including IP₃ receptor-operated Ca²⁺ stores, mitochondria, and channels in the plasma membrane, and we referred to this complex as the "pacemaker unit" (26). Ca²⁺ handling in pacemaker units controls the activation of the pacemaker conductance in the plasma membrane. Cells may have many pacemaker units, and activation of currents in domains of plasma membrane associated with pacemaker units is stochastic. Randomly occurring unitary currents result from a multitude of pacemaker units within an ICC network in the absence of a mechanism to coordinate the discharge of the pacemaker units. Depolarization caused by unitary currents activates voltage-dependent Ca²⁺ entry via a dihydropyridine-resistant Ca²⁺ conductance expressed by ICC (16, 21). Ca²⁺ entry entrains pacemaker activity by coordinating release of Ca²⁺ from IP₃ receptor-operated stores. In the present study, we have found that pacemaker activity became uncoordinated by blocking dihydropyridine-resistant Ca²⁺ entry, and this led to slowed or irregular frequencies and reduced propagation velocities. The same responses to conditions that block voltage-dependent Ca²⁺ entry have been noted in intact muscle strips (40) and in ICC-MY (17, 18). When one considers the body of observations regarding the dependence of slow wave propagation on voltage-dependent, dihydropyridine-resistant Ca²⁺ entry, i.e., the expression of dihydropyridine-resistant Ca²⁺ channels in ICC-MY, reduction in slow wave upstroke velocity when this conductance is blocked or the gradient for Ca²⁺ is reduced, slowing of pacemaker frequency and conduction velocity when Ca²⁺ entry is reduced, and the emergence of unitary potentials (18), showing loss of coordination of pacemaker units when Ca²⁺ entry is blocked, the data support the conclusion that voltage-dependent Ca²⁺ entry provides the mechanism that "couples" or entrains the intrinsic oscillator activities of pacemaker units in ICC-MY networks. The activation of voltage-dependent ion channels and current influx through these channels is highly dependent upon membrane properties, channel availability, and ionic gradients. Thus we suggest that models of slow wave generation and propagation should contain parameters for features such as 1) availability of voltage-dependent Ca²⁺ channels, 2) cable properties of ICC networks, 3) driving forces for ion species, and 4) low-resistance cell-to-cell connectivity in addition to the central cellular pacemaker mechanism and nonspecific coupling parameters.

Active propagation of pacemaker activity in GI muscles occurs in ICC networks because smooth muscle cells have no mechanism to generate or regenerate slow waves (see Ref. 13). In smooth muscle strips and intact GI organs, slow wave propagation is anisotropic; propagation occurs more rapidly around the bowel than in the longitudinal axis. Examples of propagation velocities measured with surface electrodes or intracellular surface of the feline colon are the longitudinal axis = 2.9 mm/s and the circular axis = 25 mm/s (2). The submucosal surface of canine colon was measured as follows: longitudinal axis, 6 mm/s; circular axis, 17 mm/s (27); canine stomach longitudinal axis, 11.2 mm/s; and canine circular axis, 22.7 mm/s (1). Networks of ICC-MY show no particular directional orientation (see Ref. 39 and images in this study); processes project to other cells in the network without apparent structural anisotrophy. Thus at present it is unclear how the circular propagation can be 1 order of magnitude greater than propagation in the longitudinal axis. In the present experiments, the CM was intact but we removed most of the LM layer. Under these conditions, propagation was constant and isotropic within the plane of the myenteric plexus. Thus propagation within ICC networks does not appear to account for the anisotropic propagation of slow waves observed in intact muscle strips and organs. Anisotropic propagation appears to require coupling with the orthogonal muscle fibers of the CM and LM layers. More experiments using exogenous pacing of activity and specific recording of activity in strips oriented in the longitudinal and circular axes are needed to determine the basis of slow wave anisotropy.

	GRANTS

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This study was funded by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-41315 and R01 DK-45713 (to T. K. Smith) and by National Institutes of Health (NIH) Grant P20 RR-18751 (to N. J. Spencer). Laboratory imaging was supported by NIH Grant P20 RR-18751.

	ACKNOWLEDGMENTS

 
Present address for K. J. Park: Department of Surgery, Seoul National University College of Medicine, Seoul 110-744, Korea.

	FOOTNOTES

 

Address for reprint requests and other correspondence: K. M. Sanders, Dept. of Physiology and Cell Biology, Univ. of Nevada School of Medicine, Reno, NV 89557 (e-mail: kent{at}unr.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^* K. J. Park, G. W. Hennig, and H.-T. Lee contributed equally to this work.

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