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Journal of Physiology (2001), 537.1, pp. 237-250
© Copyright 2001 The Physiological Society
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ABSTRACT |
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INTRODUCTION |
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Interstitial cells of Cajal (ICC) are found at specific locations within the tunica muscularis of the gastrointestinal (GI) tract. Studies performed on tissues of the mouse and guinea-pig have suggested that ICC in different anatomical locations have discrete physiological roles. Studies in the mouse have been aided by the fact that c-kit and stem cell factor mutant animals fail to develop certain types of ICC, and specific functional losses have been observed in these animals (Ward et al. 1994, 1995; Huizinga et al. 1995; Burns et al. 1996). For example, when ICC in the myenteric region of the small intestine (IC-MY) are lost, slow wave activity is not present, suggesting that IC-MY are pacemaker cells (Ward et al. 1994; Huizinga et al. 1995). When intramuscular ICC of the stomach and lower oesophageal and pyloric sphincters are lost, neural inputs from the enteric nervous system are greatly reduced, suggesting these cells are important mediators of neurotransmission (Burns et al. 1996; Ward et al. 1998, 2000a). Studies with neutralizing antibodies to Kit protein have supported the idea that IC-MY are pacemaker cells (Torihashi et al. 1995) and demonstrated that these cells are also needed for active propagation of slow waves in the small bowel and stomach (Ordog et al. 1999). Thus, a picture has emerged regarding the functional significance of ICC in the GI tract, and these studies have suggested that a 'division of labour' exists between pacemaker ICC (IC-MY) and ICC involved in neurotransmission (IC-IM in the stomach and IC-DMP in the small intestine: see Sanders et al. 1999).
The concept that electrical slow waves originate in ICC, actively propagate in ICC, and passively spread into electrically coupled smooth muscle cells is supported by studies showing that pacemaker activity can be recorded from isolated ICC but not from isolated smooth muscle cells (see Horowitz et al. 1999). Furthermore, when regions of the muscle with pacemaker ICC are removed by dissection, slow waves decay in a manner suggestive of electrotonic (i.e. passive) conduction (Sanders et al. 1990). These observations suggested that ICC have the ability to generate and regenerate slow waves, but smooth muscle cells do not share these mechanisms (Horowitz et al. 1999). This organization seems plausible for thin-walled organs as in the mouse, but it is unclear how ICC distributed solely within a thin surface of pacemaker cells (e.g. the myenteric region between circular and longitudinal muscle layers or the submucosal surface of the circular muscle layer in the colon) could generate enough current to activate voltage-dependent responses in the smooth muscle syncytium of thicker-walled organs of humans and large animals.
Previous studies have suggested that propagation of pacemaker activity is active in thicker-walled gastric muscles. Bauer and co-workers (1985a,b) recorded with intracellular electrodes from cells within the myenteric half and submucosal half of the circular muscle layer of canine antrum. These studies demonstrated slow wave propagation in both regions of the muscle. In studies of the canine small intestine Jimenez and co-workers (1996) observed that separation of the region near the deep muscular plexus from the myenteric half of the circular muscle layer did not block rhythmic electrical activity. These data suggest that cells other than IC-MY can provide pacemaker activity. Finally, we have previously shown that functional pacemaker-like ICC in the canine colon infiltrate septa between muscle layers well into the thickness of the circular muscle layer (see Ward & Sanders, 1990). 'Septal' ICC might provide a pathway for active propagation of slow waves deep within the muscularis and thereby increase transmission of depolarization to the smooth muscle cells.
In the present study we used the canine gastric antrum to investigate the populations of ICC within a thick-walled stomach and to characterize the relationships between ICC, smooth muscle cells and enteric neurons. Four basic classes of ICC were identified, and septal ICC (i.e. ICC between bundles of muscle fibres) were abundant. Intracellular microelectrode recordings were used to determine whether sites other than the myenteric region were capable of generating and propagating slow waves in the antral wall. The data suggest that while cells in the myenteric region provide the dominant pacemaker activity in intact antral muscles, cells within the interior of the circular muscle layer are also capable of pacemaker activity. Cells within the interior of the circular muscle layer can also be paced by extrinsic stimuli and slow waves of normal amplitude were recorded many millimetres from the stimulus. These observations suggest that there are pathways for active propagation throughout the antral tunica muscularis.
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METHODS |
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Mongrel dogs of both sexes were killed with sodium pentobarbital (100 mg kg-1). After opening the abdomen, the entire stomach, including portions of the oesophagus and duodenum, was removed and placed in a bath of oxygenated Krebs-Ringer-bicarbonate solution (KRB). For morphological and electrophysiological studies, sheets of muscularis were dissected from the ventral surface of the stomach, 7-9 cm proximal to the pyloric sphincter. Tissues were pinned to the silicon (Sylgard elastomer; Dow Corning Corp., Midland, MI, USA) base of a dissecting dish and the mucosa and inner aspect of the submucosa removed. The use and treatment of animals was approved by the Animal Use and Care Committee at the University of Nevada.
Electron microscopy
Strips of gastric antrum were placed in a fixative containing 4 % paraformaldehyde and 3 % glutaraldehyde in 0.1 M phosphate buffer (PB; pH 7.4) for 2 h at room temperature. Tissues were rinsed with the same buffer and post-fixed with 1 % osmium tetroxide for 2 h at 4 °C. Tissues were further processed for electron microscopy as previously described (Torihashi et al. 1993), and examined using a Philips CM10 transmission electron microscope.
Immunohistochemistry
Gastric antrums were isolated in a manner similar to that described above. Tissues were placed in a dissecting dish and stretched to 110 % of the original length and width prior to the removal of the mucosa by sharp dissection. The remaining tunica muscularis was subsequently fixed in ice-cold paraformaldehyde (4 % w/v in 0.1 M PB) at 4 °C for 20-30 min. Following fixation, tissues were washed for 30 min in phosphate-buffered saline (PBS, 0.05 M, pH 7.4). Immunohistochemical studies were performed on tissues that were dehydrated in graded sucrose solutions, embedded in Tissue Tek (Miles, IL, USA), and frozen in liquid nitrogen. Tissues were subsequently preincubated in bovine serum albumin for 1 h (1 % in 0.1 M PBS) before being incubated with a rabbit polyclonal antibody raised against human Kit protein (CD117; Dako, Ltd, Kyoto, Japan; 1:50 dilution in 0.1 M PBS with 0.5 % Triton X-100) at 4 °C overnight. Immunoreactivity was detected with fluorescein isothiocyanate (FITC)- or Texas-Red-conjugated secondary antibody (goat anti-rabbit 1:100, 1 h, room temperature; Vector Laboratories Inc., Burlingame, CA, USA). Control tissues were prepared in a similar manner, omitting either primary or secondary antibody from the incubation solution. Whole mount tissues were labelled with Kit antibody using the same method as outlined above. After fixation tissues were washed in PBS for 4 h (4 
1 h), and slivers of muscle were dissected from the remaining tissue. Non-specific antibody binding was reduced by incubating the tissues in 1 % bovine serum albumin for 1 h at room temperature before addition of primary antibody. Tissues and sections were examined with a Bio-Rad MRC 600 confocal microscope (Hercules, CA, USA) with an excitation wavelength appropriate for FITC (494 nm). Confocal micrographs are digital composites of Z-series scans of 10-15 optical sections through a depth of 10-15 µm. Final images were constructed with Bio-Rad 'Comos' software.
Electrophysiology
For electrophysiological experiments sheets of muscularis were dissected from the ventral surface of the stomach, 7-9 cm proximal to the pyloric sphincter. Tissues were pinned to the silicon (Sylgard elastomer) base of a dissecting dish and the mucosa and inner aspect of the submucosa removed. Strips of muscle (1 mm 25 mm) through the entire muscularis were cut parallel to the circular muscle fibres. The longitudinal layer was left attached to these strips of circular muscle since slow wave activity originates at the border between the circular and longitudinal fibres (Bauer et al. 1985a). The muscle strips were transferred to an electrophysiological chamber and pinned-out in cross-section, providing a view of the entire tunica muscularis. The tunica muscularis was subsequently dissected into the layers: (i) myenteric circular muscle, which included the longitudinal muscle and the circular muscle layer adjacent to the myenteric plexus, (ii) interior circular muscle, which consisted of circular muscle fibres in the central 1/3 of tissue, and (iii) submucosal circular muscle, which consisted of circular muscle adjacent to the submucosa. The circular muscle was dissected into three regions over a distance of 20 mm; the remaining 5 mm remained attached to the other regions of the tunica muscularis at one end of the preparation. In some experiments strips of interior and submucosal circular muscle were isolated from the remaining preparation. An explanation of the preparation and image of a representative preparation are shown in Fig. 4.
After dissection, the muscles were constantly perfused with warmed, oxygenated KRB. Bath temperature was monitored and maintained at 37.5 ± 0.5 °C. The muscles were allowed to equilibrate for approximately 2 h before intracellular recording was initiated. With cross-sectional preparations we were able to impale cells along the myenteric circular layer, within the interior circular muscle and along the submucosal circular muscle over distances of 20 mm. Cells were impaled with glass microelectrodes filled with 3 M KCl and having resistances ranging from 50 to 80 M
. Transmembrane potential was measured by a standard electrometer (WPI M-7000, Sarasotota, FL, USA), and outputs were displayed on an oscilloscope (Tektronix 5111, Beaverton, OR, USA). Electrical signals were recorded on videotape (Vetter Co., Rebersburg, PA, USA) and chart paper (Gould 2200, Cleveland, OH, USA).
Solutions and drugs
Muscles were maintained in KRB (37.5 ± 0.5 °C; pH 7.3-7.4) containing (mM): Na+, 137.4; K+, 5.9; Ca2+, 2.5; Mg, 1.2; Cl-, 134; HCO3-, 15.5; H2PO4-, 1.2; dextrose, 11.5; and bubbled with 97 % O2-3 % CO2. Acetylcholine, atropine sulphate and tetrodotoxin (Sigma, St Louis, MO, USA) were dissolved in distilled water to give a concentration of 0.1-0.01 M; nifedipine was dissolved in ethanol to give a concentration of 0.01 M and diluted in KRB to the stated final concentrations.
Analysis of intracellular microelectrode data
Several slow wave parameters were analysed: (i) resting membrane potential (RMP); (ii) slow wave amplitude (upstroke and plateau phases); (iii) duration (time to 90 % repolarization); and (iv) frequency. Averaged data are expressed with standard errors of the mean. Student's paired t tests were performed, and a P value of less than 0.05 was taken as indicting a statistically significant difference.
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RESULTS |
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Structure of ICC networks using immunohistochemical techniques
Gross morphological examination of cross-sections of the tunica muscularis of the canine gastric antrum revealed tissues with an average thickness of 4.55 ± 0.15 mm (n = 5). Both the circular and longitudinal muscle layers were subdivided into bundles of muscle fibres that were separated by connective tissue septa. Since ICC have been shown to generate and propagate slow waves in GI muscles, we investigated the anatomical locations and morphology of ICC networks within the tunica muscularis using antibodies raised against the Kit receptor, which has been used as a marker for ICC in several species (for review see Sanders et al. 1999). In cross-sections, Kit-like immunoreactivity (Kit-LI) revealed sub-populations of ICC at several levels within the tunica muscularis (Fig. 1). ICC were located in the region of the myenteric plexus between the circular and longitudinal muscle layers (IC-MY). These cells were observed along the longitudinal and circular aspects of myenteric ganglia (Fig. 1A). ICC were also observed within the circular and longitudinal muscle layers (IC-IM; Fig. 1B-D). IC-IM ran parallel to the longitudinal axis of the muscle fibres. Cells with Kit-LI were also found within septa that separated muscle bundles of the circular and longitudinal muscle layers (IC-SEP; Fig. 1B and C). Septa also contained collagen fibres, blood vessels and nerve trunks. IC-SEP were often closely associated with more than a single muscle bundle, suggesting these cells may interconnect adjacent bundles. Finally, Kit-positive cells were also located along the submucosal surface of the circular muscle layer (IC-SM). IC-SM had a similar morphology to IC-SEP and could represent a sub-population of IC-SEP (Fig. 1D). These four populations of cells with Kit-LI had ultrastructural characteristics that identified them as ICC (see below).
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Figure. 1. Kit-like immunoreactivity in ICC at different levels in the canine antrum A shows a cryostat section through the myenteric plexus region. IC-MY (arrows) were located around myenteric ganglia (mg) on both the longitudinal (lm) and circular muscle (cm) aspects of ganglia. ICC were found within the circular and longitudinal muscle layers (arrowheads; IC-IM). B shows IC-IM within the circular and longitudinal muscle layers (arrowheads). IC-IM ran parallel to the long axis of the smooth muscle cells in both layers. C, ICC also populated septa (s; IC-SEP; arrows) that separated the circular muscle layer into discrete bundles. IC-SEP transversed circular muscle bundles and were often associated with more than a single muscle bundle. D shows ICC located along the submucosal surface of the circular muscle layer (IC-SM; arrows). These cells formed a network and occasionally bridged two or more circular muscle bundles. IC-IM (arrowheads) are denoted. E-H show whole mount preparations of canine gastric antrum. E shows IC-MY (arrows). These cells formed an anastomosing network of cells with short interconnecting processes. F shows IC-SM (arrowheads). IC-SM were similar in structure to IC-SEP and processes that extended from the main axis of the cell body formed a interconnecting network with adjacent IC-SM. G and H are montages of circular and longitudinal muscle slivers, respectively. G reveals circular IC-IM located within smooth muscle bundles (arrowheads). Occasional ICC (IC-SEP; arrows) were also observed running along the outside of muscle bundles and occasionally crossed septa to interconnect adjacent smooth muscle bundles. IC-SEP were also bi-polar with occasional processes extending out to adjacent ICC. H shows longitudinal IC-IM, which were typically spindle-shaped with occasional projections extending perpendicular from bi-polar processes and had a similar morphology to circular IC-IM. These projections formed contacts with adjacent ICC to form a 3-dimensional network within the longitudinal muscle layer. Scale bars are indicated in each panel. | ||
The structure of ICC in the antrum was viewed in whole mount preparations or slivers of muscularis cut along the long axis of the muscle fibres. IC-MY formed an anastomosing network of cells that were often dense around myenteric ganglia (Fig. 1E). IC-MY did not have obvious directional orientation and appeared to project randomly in several directions. Cell processes formed connections with adjacent IC-MY. IC-SM appeared bi-polar and ran in the long axis of the circular muscle layer (Fig. 1F). These cells were often grouped together and interconnected with adjacent IC-SM. IC-IM within the circular and longitudinal muscle layers were bi-polar in shape and ran in the longitudinal axis of the muscle layers (Fig. 1G and H). Occasional fine processes extended perpendicular from the long axis of IC-IM to form apparent contacts with the processes and cell bodies of adjacent IC-IM. There were also contact points between IC-IM at the ends of cells. IC-SEP had a similar morphology to IC-IM but were distinguished by the fact that they ran along the surfaces of muscle bundles, and groups of interconnected IC-SEP formed contacts between bundles of circular muscle cells (Fig. 1G). Since the morphologies of IC-SEP and IC-SM were similar and both groups of cells lay along the surface of circular muscle bundles, it was impossible to determine whether these were actually discrete types of ICC.
Ultrastructural characteristics of cells in the spaces populated by cells with Kit-LI
Transmission electron microscopy was performed on tissue samples to study the ultrastructure of cells in the regions populated with cells with Kit-LI. IC-MY, located between the circular and longitudinal muscle layers, had numerous mitochondria and an electron-dense cytoplasm (Fig. 2A and B). These cells contained extensive endoplasmic reticulum and numerous free ribosomes (Fig. 2, inset in A and C). Caveolae and a distinct basal lamina (Fig. 2A inset) distinguished these cells from macrophages and fibroblasts that were also observed in the myenteric plexus region. IC-MY formed gap junctions with each other (Fig. 2C) and with neighbouring smooth muscle cells (Fig. 2B).
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Figure 2. Ultrastructure of IC-MY in the canine antrum A shows two IC-MY (IC) located near circular muscle cells (CM). IC-MY possessed many mitochondria compared to neighbouring circular smooth muscle cells (see also inset). Inset in A, caveolae and a distinct basal lamina were also present. B shows an IC-MY (IC) forming a close contact with a neighbouring smooth muscle cell (arrow). The inset shows the region at higher magnification revealing that the close contact is a gap junction between the IC-MY and the smooth muscle cell. C shows communication between IC-MY. Both cells have an abundance of mitochondria, extensive endoplasmic reticulum and numerous free ribosomes. A gap junction can be seen between the two IC-MY (arrow and inset). Scale bars are as indicated in each panel and represent 0.1 µm in insets in B and C. | ||
IC-IM were easily distinguished from neighbouring smooth muscle cells. IC-IM contained reduced numbers of myofilaments and lacked dense bodies (Fig. 3A-C). IC-IM contained many mitochondria and an abundance of rough endoplasmic recticulum (Fig. 3B). Caveolae were associated with the plasma membrane (Fig. 3A and B). IC-IM formed gap junctions with other IC-IM and with neighbouring smooth muscle cells (Fig. 3A (main panel and inset) and B). IC-IM of the longitudinal muscle layer displayed essentially the same ultrastructural features as circular muscle IC-IM, including numerous caveolae along the plasma membrane and a distinct basal lamina (Fig. 3C).
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Figure 3. Ultrastructure of IC-IM, IC-SEP and IC-SM A shows an IC-IM within the circular muscle layer (IC). IC-IM were distinct from neighbouring smooth muscle cells and contained a reduced filament content and lacked dense bodies. IC-IM formed multiple close associations with neighbouring smooth muscle cells (arrowheads). A distinct gap junction between the IC-IM (IC) and a neighbouring circular smooth muscle cell is indicated by the arrow and this region is shown at a higher magnification in the inset. IC-IM were also closely associated and formed gap junctions with each other (arrows). These cells contained many mitochondria, caveolae, rough and smooth ER and free ribosomes (B). IC-IM were also located in the longitudinal muscle layer (C). These cells had similar ultrastructural features to circular muscle IC-IM and possessed caveolae and a distinct basal lamina. D shows an IC-SEP and processes (*) at the interface between two circular muscle bundles. IC-SEP possessed many mitochondria, caveolae and a continuous basal lamina. Varicose nerve fibres (N) are closely associated with the IC-SEP. E and F show IC-SM at the submucosal surface of the circular muscle layer. IC-SM contained clusters of mitochondria and caveolae along the plasma membrane. A gap junction (arrow) between an IC-SM and a neighbouring smooth muscle cell (CM) is shown at higher magnification in the inset in F. IC-SM possessed numerous mitochondria, Golgi apparatus, rough and smooth endoplasmic recticulum and many free ribosomes. Numerous caveolae were present along the plasma membrane and a continuous basal lamina was observed. Scale bars are as indicated in each panel and represent 0.1 µm in insets in A and F. | ||
Immunofluorescent images showed that cells with Kit-LI also lined septae between muscle bundles. Cells with morphologies similar to IC-MY and IC-IM were readily apparent in septal spaces. IC-SEP possessed many mitochondria and an abundance of rough endoplasmic recticulum (Fig. 3D). Caveolae and a continuous basal lamina were also associated with the plasma membrane (Fig. 3D). Varicose nerve fibres containing large, dense-core and small clear vesicles were closely associated with IC-SEP (Fig. 3D). IC-SEP also formed gap junctions with each other and with neighbouring smooth muscle cells (not shown), suggesting that these cells could serve as electrical bridges between muscle bundles.
Cells with Kit-LI were also found along the submucosal surface of the circular muscle layer (IC-SM) and had ultrastructural properties characteristic of ICC (Fig. 3E and F). The cytoplasm of these cells contained an abundance of mitochondria, Golgi apparatus, rough and smooth endoplasmic recticulum and many free ribosomes (Fig. 3E and F). IC-SM were often observed near nerve bundles and formed gap junctions with each other IC-SM and with neighbouring smooth muscle cells (Fig. 3F, main panel and inset).
Locations of electrical pacemakers in gastric muscles
We performed studies to determine whether sites of pacemaker activity other than the myenteric plexus region exist within the circular muscle and how electrical events, generated at a given point, might propagate throughout the circular muscle layer. Cross-sectional muscle preparations were cut and impalements were made at various levels through the circular muscle layer. Near the myenteric surface, cells had resting membrane potentials (defined as the most negative potential between slow waves) of -74 ± 1.0 mV, and slow waves (42 ± 1.1 mV in amplitude and 7.1 ± 0.3 s in duration) occurred spontaneously at a frequency of 1.8 ± 0.08 cycles min-1 (Fig. 4A; n = 31 animals). Cells 50 % through the thickness of the circular layer had membrane potentials averaging -71 ± 1.8 mV, and this was not significantly different from myenteric cells (n = 16; P > 0.05). Slow waves were smaller in amplitude in cells impaled halfway through the circular muscle. The upstroke amplitude averaged 36 ± 2.1 mV (P < 0.01 compared to myenteric slow waves; n = 16 animals). Slow wave duration (7.7 ± 0.6 s; P = 0.406) and frequency (1.7 ± 0.12; P = 0.452) were not significantly different from those in myenteric cells. Typical recordings from this region are shown in Fig. 4F.
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Figure 4. Intracellular electrical recordings from different regions of the tunica muscularis The upper panel shows the muscle preparation used to examine the electrical activities of different regions of the circular muscle layer (myenteric, interior and submucosal regions). In the example in Fig. 4 the strips were left connected to an intact segment of the entire muscularis. In other studies (see later figures) cuts were made along the line denoted (white dashed line) to separate the 3 regions. Electrical recordings were made from each region at distances up to 20 mm from the intact portion of muscularis to determine the spread of slow waves. Spontaneous slow waves at similar frequencies were recorded at all sites along the myenteric surface (A-E), within the interior circular muscles (F-J) and in the submucosal muscle (K-O). Summary data of slow wave parameters from a series of recordings from preparations of this type are given in Table 1. Scale bar in upper panel = 5 mm. | ||
The biggest differences were observed in cells along the submucosal surface of the circular muscle layer. Resting membrane potentials were more depolarized (e.g. -64 ± 2.6 mV; P = 0.001; n = 13 animals) compared to cells along the myenteric surface. Slow wave amplitude was also significantly reduced (24 ± 2.6 mV; P < 0.001) compared to myenteric cells. Slow wave duration (7.8 ± 0.7 s) was not significantly different (see Fig. 4K). Slow waves recorded from cells near the myenteric and submucosal surfaces are similar to previously reported activities of these regions (Bauer et al. 1985b).
In mice, IC-MY appear to be necessary for slow wave propagation in gastric muscles (Ordog et al. 1999). We tested this hypothesis in the thicker-walled canine antrum by making longitudinal cuts in the circular muscle layer, creating 3 muscle strips of myenteric, interior and submucosal circular muscle (see image in Fig. 4). These strips were left attached to an intact portion of muscularis to maintain electrical contact with the dominant pacemaker region near the myenteric plexus (see Bauer et al. 1985a). Impalements were made at 5 mm intervals along the myenteric, interior and submucosal muscle strips from 44 animals. Electrical slow waves of similar amplitude and duration were recorded at all sites within a given region of muscle (Fig. 4 and summary data in Table 1). In order to complete these long experiments consisting of multiple impalements, relatively short periods of recording at each site were performed (i.e. approximately 5 min at each site). The normal variability in slow wave frequency in antral muscles made it difficult to accurately compare frequencies in these experiments, but it was apparent that slow wave frequency was relatively constant at all sites of recording. Maintenance of normal frequency might have resulted if the myenteric pacemaker continued to pace the entire muscle preparation. For this to have been true in the preparations we used, slow waves generated by myenteric pacemaker cells would have had to actively propagate down the long stretches (> 20 mm) of interior and submucosal muscle strips that were separated from the myenteric region of muscle (see image in Fig. 4).
The dominance of the myenteric pacemaker was demonstrated by creating separated strips of myenteric, interior and submucosal circular muscle by making the cut denoted by the white dashed line superimposed on the image in Fig. 4. Impalements were made from each region before and after the cut was made. Separation of the three regions did not appear to inordinately damage the muscle strips (Fig. 5). For example, the resting membrane potential of interior muscle cells 15 mm from the site of separation averaged -67 ± 2.2 mV before the cut and -67 ± 2.0 mV (P > 0.05) after dissection of the interior strips (n = 11). Separation of the strips from the myenteric region caused electrical quiescence in 9 of 11 interior muscle strips (Fig. 5B). However, in 2 interior muscle preparations spontaneous slow wave activity of relatively normal amplitude (i.e. 31 mV) was observed, although the frequency of these events was greatly reduced (Fig. 5C). In these recordings there were quiescent periods lasting up to 30 min before a slow wave occurred in the interior circular muscle. Recordings from cells in separated submucosal strips also demonstrated electrical quiescence in the majority of preparations. Slow waves in myenteric strips were similar to intact muscles (not shown). Thus continuity with the myenteric region of muscle is necessary to maintain normal slow wave frequency throughout the thickness of the muscularis, but interior and submucosal muscles separated from myenteric pacemakers retain the ability to generate electrical slow waves. Thus pacemaker activity is not restricted to the myenteric region (i.e. IC-MY) in the canine gastric antrum.
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Figure 5. Electrical activity in interior circular muscle before and after isolation from the intact muscularis A shows electrical activity in an interior circular muscle strip 15 mm from the intact region of muscularis in a preparation like that shown in Fig. 4 (recording made from point I in Fig. 4). B shows loss of spontaneous slow wave activity (observed in 9 of 11 preparations) after the interior circular muscle was isolated from the intact region of muscularis. In 2 preparations, spontaneous electrical activity was recorded in interior circular muscle strips after isolation from the intact muscularis (C). | ||
Stimulation of slow waves by extrinsic stimuli
The dramatic reduction in slow wave frequency in separated interior and submucosal circular muscles as compared to intact strips of muscularis confirms that the dominant pacemaker lies within the myenteric region, as suggested by Bauer et al. (1985a). The interior and submucosal strips appear to lack pacemakers active enough to generate normal antral slow wave rhythms but have the capability of propagating slow waves when suitably paced by myenteric pacemakers. To investigate this possibility we performed pacing experiments to determine the ability of each portion of the muscle layer to respond to extrinsic pacing.
The first experiments were performed on preparations like the one shown in Fig. 4. Simulating wires were placed at the end of the intact segment of muscularis and a recording electrode was used to impale cells in the myenteric, interior and submucosal regions 20 mm from the site of stimulation. Electrical field stimulation (EFS; single pulses, 0.5-5 ms in duration, 15 V) evoked slow waves in each of the three regions of the circular muscle. When EFS was delivered too soon between slow waves either no response occurred or a premature slow wave of reduced amplitude was evoked due to the refractory properties of these muscles (see Publicover & Sanders, 1986). However, pacing the circular muscle layer at a constant frequency of 0.05 Hz resulted in slow wave responses at 3 cycles min-1 with high fidelity in each region of muscle (Fig. 6). Generation and propagation of evoked slow waves from the site of stimulation into each region were resistant to atropine (1.0 µM) and TTX (0.3 µM) (n = 3).
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Figure 6. Spontaneous and paced slow waves in myenteric, interior and submucosal circular muscle strips (preparations as shown in Fig. 4) A and B are spontaneous and paced slow waves recorded from myenteric strips. C-F are recordings from interior and submucosal circular muscle strips, respectively. All recordings were made 20 mm from the intact portion of muscularis and 25 mm from the site of EFS. | ||
We attempted to evoke slow waves from interior circular muscle strips isolated from the remainder of the muscularis. First, spontaneous slow waves were recorded from the interior circular muscle region in a preparation like that shown in Fig. 4 (Fig. 7A). Then slow waves were paced at 0.05 Hz (Fig. 7B). Then the interior region was separated from the intact segment of muscularis and EFS (0.5-10 ms; 0.05 Hz) was delivered via stimulating wires placed at one end of the separated interior muscle strips. Quiescent muscle strips (Fig. 7C) could be readily paced and generated slow waves of relatively normal characteristics averaging 26 ± 1.9 mV in amplitude and 4.4 ± 0.4 s in duration. There was a one-to-one relationship between stimuli and slow waves in these separated interior muscles (n = 9; Fig. 7D).
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Figure 7. Spontaneous and paced slow waves in interior circular muscle when this region was attached (A and B) and isolated (C and D) from the intact region of the muscularis A shows normal slow wave activity and frequency. B, the frequency could be increased by electrical pacing with EFS. Then the interior muscle strip was separated by sharp dissection from the intact region of the muscularis (as shown by the white dashed line in the image in Fig. 4). This caused loss of slow wave activity (C), but pacing of the isolated strip of interior circular muscle restored slow wave activity (D). | ||
We sought to enhance the excitability of interior muscle pacemakers in quiescent strips by superfusing the muscles with solutions containing acetylcholine (ACh). ACh (0.3 µM) caused membrane depolarization from -66.0 ± 1 to -63 ± 1.8 mV and induced regular slow waves with an upstroke amplitude of 23.0 ± 2.6 mV, a plateau amplitude of 17.0 ± 2.7 and a duration of 6.9 ± 0.7 s (Fig. 8). The average frequency of slow waves evoked by ACh was 3.6 ± 0.4 cycles min-1 (n = 6). Slow waves evoked by ACh were not blocked by nifedipine (n = 4).
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Figure 8. Slow wave activity was restored in isolated interior muscle strips by cholinergic stimulation A shows lack of slow wave activity in an isolated interior muscle strip. B, ACh (0.3 µM) produced slight membrane depolarization and evoked slow wave activity in the same muscle strip. C, washout of ACh restored electrical quiescence. Traces in A-C are excerpts from a continuous recording from the same cell. | ||
Previous studies have shown that electrical slow waves in gastric antral muscles are reduced but not blocked by dihydropyridines (Ozaki et al. 1991; Dickens et al. 1999). We tested whether events recorded from interior muscle strips met this criterion. Spontaneous and paced slow wave activity was recorded from myenteric circular muscle cells and cells within interior circular muscle strips. Nifedipine (1 µM) had no effect on resting membrane potential along the myenteric surface (-70.0 ± 1.6 mV versus -70.0 ± 2.6 before and after nifedipine; P > 0.05; n = 8), and did not significantly decrease the upstroke or plateau phase of slow waves (upstroke and plateau amplitudes were, respectively, 42.4 ± 1.7 mV and 27.6 ± 1.6 mV in control and 39.6 ± 2.2 mV and 24.2 ± 3.1 mV in the presence of nifedipine; see Fig. 9A-D). In interior circular muscle, nifedipine did not affect the membrane potential (-70.5 ± 3.6 mV versus -70.8 ± 3.5 before and after nifedipine; P > 0.05) and did not reduce the upstroke and plateau amplitudes of slow waves (i.e. from 30.8 ± 2.3 mV and 19.3 ± 1.5 mV to 29.5 ± 3.7 mV and 15.3 ± 2.0 mV, respectively; see Fig. 9E-H). In interior muscles separated from the remainder of the muscularis, nifedipine (1 µM) had no significant effect on slow waves evoked by EFS (Fig. 9J).
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Figure 9. Nifedipine did not block spontaneous and evoked slow waves A and B show spontaneous slow waves recorded from the myenteric region of the circular muscle layer before and after addition of nifedipine (1 µM) for 20 min. C and D show paced slow wave activity before and after nifedipine. E-H show the effects of nifedipine on spontaneous and evoked slow waves recorded from an interior circular muscle strip attached to the intact region of muscularis (as in Fig. 4). Nifedipine partially reduced the amplitude of the plateau phase and reduced the duration of slow waves. Nifedipine had no effect on resting potential (I) and did not block slow waves evoked in isolated interior circular muscle by EFS (J). | ||
Propagation velocity in myenteric versus interior circular muscle strips
We compared the propagation velocities of slow waves in myenteric and interior muscle strips. Figure 10 shows the rate of spread of slow waves along the myenteric surface and within interior muscle strips. The time required for spread of electrical activity from the site of stimulation to various recording sites was plotted as a function of distance. Linear regression analysis resulted in slopes of 0.0602 ± 0.008 (n = 5; r 2 = 0.954) and 0.052 ± 0.002 (n = 5; r 2 = 0.9951) for myenteric and interior strips, respectively. The inverse of the slopes gave propagation velocities of 16.6 and 19.2 mm s-1 in myenteric and interior muscle strips.
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Figure 10. Propagation of slow waves in myenteric and interior muscle strips Slow waves were recorded at various distances (5-25 mm) from the site of stimulation (EFS site). The latencies between the time of stimulation and the time at which slow waves occurred at the various recording sites were plotted as a function of distance from the site of stimulation. Slow waves from several recording sites are superimposed in A (myenteric muscle strip) and B (interior muscle strip). C shows a summary of slow propagation in 5 myenteric muscle strips. The data were fitted by linear regression analysis and the best line had a slope of 0.0602 ± 0.008 (n = 5; r2 = 0.954). The inverse of the slope gave a propagation velocity of 16.6 mm s-1. D shows a summary of slow wave propagation in 5 interior circular muscle strips. Linear regression analysis of the data points gave a slope of 0.052 ± 0.002 (n = 5; r 2 = 0.9951). The inverse of the slope gave a propagation velocity of 19.2 mm s-1. | ||
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DISCUSSION |
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In this study we characterized the structure of ICC and ICC networks within the canine gastric antrum using immunofluorescence (with anti-Kit antibodies as a marker for ICC) and electron microscopy. We found that ICC populate four general locations within the tunica muscularis of the canine gastric antrum: (i) ICC within the region between the circular and longitudinal muscle layers (IC-MY); (ii) intramuscular ICC that lie within bundles of circular and longitudinal muscle cells (IC-IM); (iii) ICC that lie within septa between bundles of smooth muscle cells (IC-SEP); and (iv) ICC at the submucosal surface of the circular muscle layer (IC-SM). IC-IM, IC-SEP and IC-SM were all closely associated with neural processes that were neuronal nitric oxide synthase positive (not shown), and the ultrastructural features of these cells were similar. Thus, our categorization of ICC is based on anatomical position only and should not imply differences in phenotype or function. Since ICC are known to generate and propagate electrical slow wave activity (e.g. Ward et al. 1994; Huizinga et al. 1995; Dickens et al. 1999; Ordog et al. 1999), the presence of the various classes of ICC in canine gastric antrum provides a structural basis for the functional observations we made.
Our data suggest that in addition to IC-MY some, if not all, of IC-IM, IC-SEP and IC-SM may possess the ability to generate electrical slow waves either spontaneously or in response to electrical pacing. Suzuki & Hirst (1999) and van Helden and coworkers (2000) have reported that it is possible to evoke slow wave-like events in small bundles of gastric muscle from the guinea-pig. These small strips, although likely to contain IC-IM and/or IC-SEP, were unlikely to contain IC-MY, the normal pacemaker cells in the guinea-pig stomach (Dickens et al. 1999). Our data are consistent with these findings and show that even in quiescent muscle strips lacking IC-MY, electrical current can evoke slow waves.
At the present time there is no direct evidence demonstrating that ICC generate electrical slow waves in the canine gastric antrum. Indirect evidence for this hypothesis is as follows. Simultaneous recordings from gastric ICC (IC-MY) and smooth muscle cells of the guinea-pig stomach showed that electrical slow waves occurred first in ICC and then in smooth muscle cells (Dickens et al. 1999). This suggests that a pacemaker mechanism in ICC drives slow wave activity. Murine gastric muscles made deficient in ICC with neutralizing Kit antibodies and in diabetes lose slow wave activity (Ordog et al. 1999, 2000). Isolated canine gastric antral smooth muscle cells are excitable, but they do not appear to be capable of generating slow waves (e.g. Vogalis et al. 1991). Canine gastric antral muscle cells responded to depolarization with activation of L-type Ca2+ channels (and a variety of voltage-dependent K+ channels), Ca2+ entry and contraction, but there is no evidence to suggest that the slow wave mechanism is part of the phenotype of gastric antral smooth muscle cells. It is also clear that electrical slow wave activity in the gastric antrum is not neurogenic, because application of TTX had no effect on spontaneous rhythmicity (Szurszewski, 1975; this study). The present study demonstrates that anatomical features of canine gastric ICC are similar to the ICC of mouse and guinea-pig (Burns et al. 1996, 1997). Previous studies have demonstrated autorhythmicity in isolated canine ICC (Langton et al. 1989). Taken together, these data suggest that electrical rhythmicity is likely to be a feature of ICC in the canine antrum, but further tests of the link between ICC and generation of electrical slow waves in canine tissues will await the discovery of a method to remove specific populations of ICC without affecting other cell types in the muscularis.
Specific physiological roles for gastric IC-MY and IC-IM have been determined from studies of murine and guinea-pig stomachs. In guinea-pig, gastric slow waves occurred first in IC-MY and then spread to adjacent smooth muscle cells (Dickens et al. 1999). Stomachs of wild-type mice have IC-IM in the fundus but no electrical slow waves, suggesting that IC-IM are not capable of spontaneously generating slow waves. Stomachs of W/WV mutant mice lack IC-IM, but IC-MY are found throughout the corpus and terminal stomach (Burns et al. 1996). These animals had significantly reduced neural responses (Burns et al. 1996; Ward et al. 2000a) but essentially normal slow wave activity (Burns et al. 1996). When IC-MY were lost after treatment with neutralizing Kit antibodies, slow waves were also lost, and electrical pacing failed to elicit slow waves (Ordog et al. 1999). Taken together, these observations suggest that IC-MY are necessary for generation of slow waves and serve as the dominant pacemaker cells in the stomach. IC-IM do not have the capacity to generate slow waves in the murine stomach but provide an important role in neurotransmission. Recent studies also suggest a role for IC-IM in propagation of slow waves in the mouse (Dickens et al. 2001).
The division of labour between IC-MY and IC-IM deduced from studies of the mouse may not be applicable to the canine antrum and possibly to other large mammals. We found, as suggested by previous studies (Bauer et al. 1985a), that the pacemaker in the myenteric region is dominant in intact muscles, but when sub-regions of the tunica muscularis were separated from the myenteric region, spontaneous slow wave activity persisted in some tissues. The dissected muscles contained IC-IM and IC-SEP and, in the case of strips of muscle from the submucosal portion of the circular muscle layer, IC-SM. IC-IM, IC-SM and IC-SEP had similar morphologies and relationships with neighbouring smooth muscle cells. At the present time techniques do not exist to determine whether there are phenotypic differences between IC-IM, IC-SM and IC-SEP or which cell type is responsible for the spontaneous pacemaker activity recorded from isolated strips of interior and submucosal circular muscle.
Recent studies have suggested that guinea-pig stomach (including the pylorus) may have properties similar to the dog antrum. Small bundles of guinea-pig gastric muscle lacking the myenteric border generated noisy depolarization events (spontaneous transient depolarizations; STDs) that could sum to produce 'regenerative potentials' (Suzuki & Hirst, 1999; van Helden et al. 2000). Regenerative potentials had many of the properties of the tissue response to pacemaker activity including sensitivity to caffeine, insensitivity to nifedipine and an extended period of refractoriness (Publicover & Sanders, 1986; Suzuki & Hirst, 1999; van Helden et al. 2000). These studies did not directly determine whether the regenerative potential response was a product of IC-IM (which were verified to be present in the tissue bundles by Kit immunoreactivity) or was produced by smooth muscle cells (Suzuki & Hirst, 1999); however, mutations lacking IC-IM do not generate spontaneous transient depolarizations. Studies of isolated smooth muscle cells from the guinea-pig antrum and corpus have demonstrated a transient (L-type) Ca2+ conductance in response to depolarization, but not an inward current insensitive to nifedipine (e.g. see Wade et al. 1996; Kim et al. 1997). These data, although indirect, suggest that regenerative potentials could be a response to depolarization initiated in IC-IM. However, cellular studies on IC-IM have not yet been performed to determine the nature of the ionic current that mediates regenerative potentials. An equivalent conductance may be present in IC-IM, IC-SM or IC-SEP in canine gastric muscularis, and electrical pacing of isolated interior muscle strips in our experiments may have activated this conductance.
Based on our current concept of the pacemaker mechanism in GI muscles, it is unclear how exogenous current evokes slow waves. Slow waves result from pacemaker currents that depend upon activation of a voltage-independent non-selective cation conductance (Thomsen et al. 1998; Koh et al. 1998). Studies of pacemaker ICC from the mouse small intestine have shown that gating of the pacemaker conductance is controlled by a process involving Ca2+ release by IP3 receptors in the sarcoplasmic reticulum (SR) and Ca2+ uptake by mitochondria (see Ward et al. 2000b; Suzuki et al. 2000). The current study showed that current applied to all regions of the circular muscle layer evoked slow waves. Slow waves can also be evoked in small bundles of muscle from the guinea-pig antrum and pylorus (Suzuki & Hirst, 1999; van Helden et al. 2000). These muscles appear to generate slow waves via the same IP3-receptor/mitochondrial mechanism (Ward et al. 2000b). Depolarization might activate voltage-dependent entry of Ca2+ into pacemaker cells that, in turn, might enhance the open probability of IP3 receptors and initiate the slow wave mechanism. It is also possible that depolarization might activate a voltage-dependent increase in IP3 production (see Ganitkevich & Isenberg, 1993; Mahaut-Smith et al. 1999). It should be noted that cholinergic stimulation, which is known to increase the frequency of slow waves in the gastric antrum (Szurszewski, 1975; El-Sharkawy & Szurszewski, 1978) might also have enhanced slow wave activity in antral muscle strips by increasing IP3.
Our data show that cells within the interior of the circular muscle layer of antral muscles are capable of active propagation of slow waves. When slow waves were evoked at one end of muscle strips, events of approximately the same amplitude and waveform were recorded at distances many millimetres from the stimulating electrodes. Since the length constant of canine gastric muscles in the long axis of the circular muscle fibres is only about 1.7-2.4 mm (Bauer & Sanders, 1986), one would expect that without active regeneration, slow waves would not persist over the distances tested. From our studies we cannot be sure which cells within the muscularis are responsible for active propagation, but in murine gastric muscles with missing patches of ICC, slow waves rapidly decay in amplitude away from pacemaker regions (Ordog et al. 1999). Similar decay of slow waves has been observed in muscles of the canine colon in which the submucosal pacemaker cells were removed by dissection (Sanders et al. 1990). Removal of the myenteric pacemaker in the canine gastric antrum, however, did not disrupt active propagation, suggesting that the population of ICC at the myenteric border is not critical for regeneration of slow waves. It is possible that in addition to generating slow waves, pacemaker cells within the tunica muscularis serve as an active propagation pathway that transmits slow waves to all parts of the smooth muscle syncytium.
In summary, this study expands the concept of how and where slow waves are generated in thick-walled gastric tissues. We have provided evidence that pacemakers are distributed throughout the tunica muscularis of the canine gastric antrum. While the dominant pacemaker resides in the myenteric region in a given segment of muscularis, other cells within the wall also possess the capability of generating and regenerating slow waves. Thus, slow waves are able to propagate with relatively little decrement through the syncytium of smooth muscle. Spread of the slow wave depolarization into the smooth muscle cells would tend to activate L-type Ca2+ channels (see Vogalis et al. 1991; Sims et al. 1992) and these couple excitation to contraction. The canine gastric antrum has abundant ICC that are electrically coupled to smooth muscle cells and in tight association with intramural nerve processes. There is currently no direct evidence available to demonstrate that ICC within the muscle wall (IC-IM or IC-SEP) are the source of pacemaker activity and cells responsible for active propagation of slow waves. However, smooth muscle cells do not generate slow waves and evidence from other mammalian species suggests that ICC are likely to express the molecular apparatus necessary to generate and propagate slow waves.
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Acknowledgements
This study was supported by grants from the National Institutes of Health (DK 40569 and DK 57236). The authors are grateful to the morphological core facility provided by DK 41513 for immunohistochemistry and electron microscopy. The authors are also grateful to J. Bayguinov for technical assistance with the Kit immunofluorescence studies and to Fiona Mitchell for technical assistance with electrophysiological studies.
Corresponding author
S. M. Ward: Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV 89557, USA.
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