Comparative morphology of interstitial cells of Cajal Ultrastructural characterizationкод для вставкиСкачать
MICROSCOPY RESEARCH AND TECHNIQUE 47:239–247 (1999) Gastrointestinal Peristalsis: Joint Action of Enteric Nerves, Smooth Muscle, and Interstitial Cells of Cajal JAN D. HUIZINGA* Intestinal Disease Research Programme and Department of Medicine, McMaster University, Hamilton, ON L8N 3Z5, Canada KEY WORDS ICC; pacemaker; intestinal motility; rhythmicity ABSTRACTS Peristalsis is a propulsive motor pattern orchestrated by neuronal excitation and inhibition in cooperation with intrinsic muscular control mechanisms, including those residing in interstitial cells of Cajal (ICC). Interstitial cells of Cajal form a network of cells in which electrical slow waves originate and then propagate into the musculature initiating rhythmic contractile activity upon excitaton by enteric nerves. Interstitial cells of Cajal have now been isolated and their intrinsic properties reveal the presence of rhythmic inward currents not found in smooth muscle cells. In tissues where classical slow waves are not present, enteric cholinergic excitation will evoke slow wave-like activity that forces action potentials to occur in a rhythmic manner. Intrinsic and induced slow wave activity directs many of the peristaltic motor patterns in the gut. Microsc. Res. Tech. 47:239–247, 1999. r 1999 Wiley-Liss, Inc. INTRODUCTION Peristaltic motor activity is a motor pattern orchestrated by complex sequencing of neuronal excitation and inhibition in cooperation with intrinsic muscular control mechanisms, including those residing in interstitial cells of Cajal (ICC). Peristalsis is defined as waves of contraction propagating along the gastrointestinal tract for various distances as a means of mixing and propelling its content distally. Both the type of neuronal activity and the type of intrinsic myogenic control mechanism differ widely throughout the gastrointestinal tract. Interestingly, peristalsis is often equated with the peristaltic reflex (Grider and FoxxOrenstein, 1999). The peristaltic reflex as evoked by pinching or pulling at a point along the intestine, is of obvious physiological interest but is ‘‘unlikely to occur during the normal passage of liquid contents in the small intestine. . .;thus may be rarely, if ever, activated in vivo’’ (Tonini et al., 1996). Physiological activation of peristalsis will in most cases involve the stretching of a segment of stomach, intestine, or colon and it will occur by neuronal pathways that contain additional mechanisms to those required for the ascending excitatory reflex (Tonini et al., 1996). When peristaltic motor activity occurs, in particular in the stomach and proximal small intestine, the waves of contraction always have rhythmicity to it (Fig. 1). This rhythmicity is determined by electrical slow wave activity in the musculature, referred to as pacemaker activity (Huizinga et al., 1997). This review focuses on the role of interstitial cells of Cajal in the generation of peristalsis. Interstitial cells of Cajal are highly branching cells that occur in networks associated with the plexuses of the enteric nervous system. ICC are in electrical communication with the smooth muscle cells. ICC are the cells in which pacemaker activity is initiated (Koh et al., 1998; Thomsen et al., 1998) and then, together with smooth muscle cells, generate the tissue slow wave activity. Therefore, ICC can be considered to be part of the myogenic control system. That the ICC and smooth r 1999 WILEY-LISS, INC. muscle cells originate from the same mesenchymal precursor cell supports this classification (Klüppel et al., 1998; Torihashi et al., 1997). The fact that interstitial cells of Cajal are always very intimately associated with neural structures (Berezin et al., 1988; Thuneberg, 1982) indicates interaction between the myogenic and neural control systems at the level of ICC. In fact, interstitial cells of Cajal may mediate some types of inhibitory neurotransmission (Ward et al., 1998). Therefore, ICC are a crucial link in both neural and myogenic control mechanisms for peristalsis. INTERSTITIAL CELLS OF CAJAL AND THE GENERATION OF SLOW WAVE ACTIVITY The entrance of interstitial cells of Cajal into the arena of the physiology of gastrointestinal motility was greatly facilitated by the work of Lars Thuneberg and Juri Rumessen (Thuneberg, 1982). They continued work done by Taxi and others reviewed elsewhere (Thuneberg, 1989; Thuneberg et al., 1995). Thuneberg not only introduced the ICC to investigators in this field, but also introduced the means of identifying them. Furthermore, he provided or re-introduced many theories of physiological functions of ICC. Electronmicroscopy identified the ICC as a special network of cells distinct from the enteric nervous system. Together with Faussoni-Pelligrini, hypotheses were provided on the role of several independent ICC networks and their relationship with enteric nerves and smooth muscle cells (Faussone Pellegrini et al., 1977; FaussonePellegrini, 1992). Many investigators provided evidence for a role of ICC in the generation of slow waves by selectively removing those sections of the tissue that harboured the ICC. In this way, the ICC network Contract grant sponsor: Medical Research Council of Canada. *Correspondence to: Dr. Jan D. Huizinga, McMaster University, HSC-3N5C, 1200 Main Street West, Hamilton, ON L8N 3Z5 Canada. E-mail: firstname.lastname@example.org Received 1 July 1999; accepted in revised form 22 August 1999 240 J.D. HUIZINGA pioneered by Thuneberg (Thuneberg and Peters, 1987). After a few days in culture, ICC regain a morphology that is similar to that in situ. Nevertheless, positive identification of these cells requires more than morphological similarities. Optimization of measurements of kit mRNA in single cells led to the detection of kit mRNA in most cells identified as ICC by morphological criteria (Huizinga et al., 1999a ; Thomsen et al., 1998). The cells identified as interstitial cells of Cajal produced very regular inward currents, associated with membrane potential oscillations (Fig. 2) (Lee et al., 1999; Thomsen et al., 1998). This result, recently confirmed by others (Koh et al., 1998), provided conclusive evidence that ICC are pacemaker cells generating the rhythmic electrical oscillatory activity that is determining the rhythmic contractile activity of the gut musculatures. ICC, by initiating slow wave activity and through communication with the enteric nervous system (although few mechanistic details of this are known yet), play a crucial role in many, if not all, types of peristaltic motor activity. Fig. 1. Slow wave driven peristalsis implies that ICC play a role in peristaltic motor activity. When the stomach of the mouse is filled with barium contrast fluid, peristaltic activity in the distal stomach causes emptying of the stomach and filling of the proximal small intestine. This causes activation of cholinergic nerves resulting in excitation of the musculature. This leads to contractions of short sections of the circular muscle layer (see arrowheads), which propagate in aboral direction. The frequency and propagation velocity of this motor activity are identical to the frequency and propagation velocity of the electrical slow wave activity. The direction of propagation is indicated with an arrow and the position of the pylorus with a small arrow. The frequency of slow waves is ⬃ 40 per minute in the mouse small intestine, hence three sequential contractions are seen. The moment depicted, slow waves occurred only in the contracted segments; moments later the slow waves will be positioned more distally at all three sites. The slow wave activity in the mouse has been shown to originate in interstitial cells of Cajal (Thomsen et al., 1998). See also Der-Silaphet et al. (1998). responsible for pacemaker activity was identified as being in the Auerbach plexus area in the stomach and small intestine and in the submuscular plexus area in the colon, at the border of submucosa and circular muscle (Huizinga et al., 1997; Sanders, 1996). The search for the physiological significance of ICC has made important steps forward since the discovery that ICC harbor the Kit protein (Maeda et al., 1992). Studies on mutant mice that do not express functional Kit proteins showed an absence of slow wave activity as well as an absence of several types of ICC including those responsible for pacemaking activity, as reviewed recently (Huizinga et al., 1997; Sanders, 1996). Although this evidence was compelling, it was still not clear whether the ICC played an essential role in generating the slow wave activity or whether they were the cells from which slow waves originated and, hence, truly the pacemaker cells of the gut. Such confirmation required the investigation of properties of single isolated ICC and the required isolation procedures were ELECTROPHYSIOLOGY OF THE SLOW WAVE AND OF INTERSTITIAL CELLS OF CAJAL Interstitial cells of Cajal in culture have now been shown to generate slow wave activity. Although we still know very little about the electrophysiological properties of ICC, evidence points to a nonselective cation channel that is responsible for the initiation of the slow wave and hence could be called the pacemaker channel (Koh et al., 1998; Thomsen et al., 1998). Full characterization of this channel awaits further experimentation. Is this slow wave activity in isolated ICC identical to that observed in tissue? Slow waves as observed in tissue are periodic oscillations of the cell membrane potential that are omnipresent and have characteristic frequencies in each organ in each animal, between 3 and 50 cycles per minute. In ICC isolated from the mouse small intestine, the frequency is about 10 cycles/minute at room temperature, similar to the tissue slow wave activity at that temperature. The slow waves cause alternation of brief periods of high and low excitability. The period of high excitability corresponds to the plateau phase of the slow wave. Upon excitatory stimulation, the plateau phase of the slow wave rises above threshold for activation of L-type calcium channels and, consequently, action potentials (fast membrane potentials changes, the depolarization being due to calcium influx through L-type calcium channels) are generated (Huizinga et al., 1997). The calcium influx causes contraction of smooth muscle cells. Action potentials occur only superimposed on the plateau phase of the slow waves. Action potentials are a typical manifestation of smooth muscle activity and ICC may not generate them normally. Contractions in ICC are often seen to occur only in part of the cell, suggesting that contractions are mediated by local calcium release from the SR instead of through general activation of L-type calcium channels. The intensity (i.e., amplitude and frequency) of action potential generation is directly related to the force of contraction of the musculature. The slow waves determine the maximum frequency of contractions. The slow waves also determine propagation characteristics of the contrac- MECHANISMS OF PERISTALSIS 241 Fig. 2. Spontaneous rhythmic inward currents from an ICC isolated from the adult mouse small intestine. The activity was recorded at a holding potential of 0 mV, with an acquisition frequency of 66 Hz. The recording was obtained immediately after the whole cell configuration was established. The ICC had been 4 days in culture. tions. When measured in vivo, the slow wave activity is seen to propagate in an aboral direction at a velocity of approximately 2 cm/second. The reason for propagation is that the slow wave exhibits an intrinsic frequency gradient (Diamant and Bortoff, 1969; Szurszewski, 1987). That is, when pieces of tissue are taken out of an organ and examined, the slow wave frequency will be higher in the proximal part compared to the distal part. In vivo, however, because of the electrical coupling between cells in the muscle layers, the slow waves are entrained; the proximal slow waves pace the distal slow waves and hence the proximal slow waves lead. There is a certain phase lag (time lag) between the start of a proximal slow wave and the premature activation (pacing) of a distal one and hence the slow waves appear to propagate. When excited sufficiently, a slow wave is associated with circular muscle contraction. Slow waves occur virtually simultaneously at any point across the circumference of an organ and hence a ring contraction of circular muscle develops. This leads to an increase in intraluminal pressure. Because the slow waves and, therefore, the associated circular muscle contraction ring propagate distally, the (semi-) occlusion propagates distally. When the contraction is forceful enough it will push contents anally (Huizinga et al., 1997). The origin of the frequency gradient likely comes from ICC at the proximal side having some different intrinsic properties compared to ICC at distal sites. The identification of a nonselective cation channel as generating the pacemaker current is consistent with tissue data that suggested this (Liu et al., 1995; Malysz and Huizinga, 1997). The hypothesis is that rhythmic calcium release from the SR will trigger the pacemaker current. In addition we identified typical whole cell outward current profiles in ICC (Lee et al., 1999) and a beginning is made in unraveling the individual K⫹ currents. A Ba2⫹-sensitive inwardly rectifying K⫹ current in ICC that may be involved in slow wave repolarization and maintenance of a negative potential between slow waves was found by Ward and co-workers (Horowitz et al., 1999; Koh et al., 1998). Attempts have been made to characterize ICC from the dog colon (Langton et al., 1989) and L-type calcium channels as well as a 4-amino- pyridine resistant voltage activated K channel were found (Lee and Sanders, 1993). ROLE OF SLOW WAVES IN GENERATION OF ACTION POTENTIALS If action potentials were to be generated without any type of underlying depolarization, propagation of the action potentials would occur randomly in all directions. This is well demonstrated in the feline small intestine (Lammers et al., 1999) and myometrium (Lammers et al., 1994). Propagation patterns were studied by Lammers and co-workers with a battery of 240 electrodes each 1 mm apart. Propagation appeared random. In addition, in 17-day pregnant rats, marked asynchrony of bursts in different areas of the myometrium was observed suggesting non-uniform spatial and temporal distribution of excitation across the organ (Lammers et al., 1994). A major determinant of propagation patterns was the refractory period following an action potential, which could stop colliding action potentials (Lammers et al., 1994). The generation of an action potential results in a brief increase in intracellular calcium, a propagating action potential results in a wave of high intracellular calcium apparently propagating from one cell to another. This was demonstrated in the longitudinal muscle of the guinea pig colon (Stevens et al., 1999). The calcium waves propagated from cell to cell in a random fashion. Propagation was restricted by refractory periods associated with the action potentials. The guinea pig colon does not have regular slow wave activity and it will be interesting to see how calcium waves are modified in tissue with regular slow wave activity. In the myometrium, synchronization may be improved during expulsion of the fetus by enhanced synchronization due to increased numbers of gap junctions and oxytocin receptors and uniform excitation by oxytocin (Huizinga et al., 1992). Because action potentials propagate over limited distances and propagate in random directions, slow waves have evolved to control tissue excitability temporally and spatially. This forces action potential generation into rhythmic patterns, furthermore leading to apparent propagation of action potential activity in 242 J.D. HUIZINGA Fig. 3. Distention results in periodic neural excitation inducing slow wave driven peristalsis in a segment of the mouse small intestine in vitro. Top three traces: electrical activity from 3 different sites 1 cm apart, recorded simultaneously. Bottom three traces: intraluminal pressure changes recorded simultaneously at intraluminal sites of the electrodes. At bottom, pulsatile outflow was recorded (j). The segment of intestine was distended by setting the intraluminal pressure at 4 cm H2O. Action potentials occurred on 9–11 slow waves followed by 2–6 slow waves without action potentials. The accompanying intraluminal pressure changes were accordingly periodic. Outflow occurred with high amplitude propagating pressure waves (j). The direction and speed of propagation were verified by visual inspection of propagation of indentations in the muscle wall due to the circular muscle contractions. Reproduced from Huizinga et al. (1998) with permission of the publisher. aboral direction. In various tissues, the initiation of the slow wave is an exclusive property of interstitial cells of Cajal. In other tissues, slow wave-like activity develops serving the same function but with smooth muscle cells and enteric nerves playing a critical role in its initiation (see below). It has always been seen as problematic that a small group of cells such as the ICC can critically influence a much larger group of smooth muscle cells. The idea is that any current generated by the ICC would dissipate into the large mass of cells to which they are coupled. The explanation has to lie in a special nature of the three-dimensional coupling characteristics between ICC and smooth muscle cells (Publicover, 1995). It may also be that close apposition contacts or gap junctions that are coupling ICC to smooth muscle cells are of a special nature. Dye coupling has shown that dye spreads easily from ICC to smooth muscle cells but not as readily from smooth muscle cells to ICC, suggesting a rectifying type of junctional coupling (Liu et al., 1998). A difference in coupling between smooth muscle cells and between ICC and smooth muscle cells was also observed when studying transfer of electrical current (Dickens et al., 1999). DISTENTION-INDUCED PERISTALSIS Motor Patterns Determined by Cholinergic Excitation and Slow Wave Activity It is likely that most patterns of peristalsis are induced by some form of distention of the bowel wall. Weems and Seygal (1981) showed that a segment of cat ileum subjected to intraluminal pressures from 5–10 cm H2O developed periodic motor activity with time intervals of ⬃ 8 minutes. These bursts of motor activity led to an outflow of contents in an aboral direction. The bursts of motor activity were abolished by tetrodotoxin and atropine and, therefore, initiated, at least in part, by activity of intrinsic cholinergic neurons. This excitation moves along the intestine directed by the enteric nervous system. When cholinergic nerves excite the musculature, intrinsic slow waves generated by the musculature surpass the threshold for action potential generation due to depolarization, and action potentials occur, superimposed on each slow wave. As a consequence, propagating rhythmic contractile activity at the slow wave frequency occurs. In addition, in vivo, such slow wave-driven peristalsis was shown to occur after distention by content received from the stomach (Fig. 1) (Der-Silaphet et al., 1998). The associations between intraluminal pressure, electrical slow wave activity, and pulsatile outflow were demonstrated in an in vitro model of distention (Fig. 3). The intraluminal pressure was allowed to rise to between 2 and 4 cm H2O in a segment of proximal intestine of the mouse (Huizinga et al., 1998). A bursting pattern of action potential generation was elicited that was sensitive to TTX. Action potentials occurred on several sequential slow waves, alternating with slow waves without action potentials. Slow waves with superimposed action potentials were associated with increases in intraluminal pressure (Fig. 3). The slow MECHANISMS OF PERISTALSIS waves and the transient increases in intraluminal pressure propagated along the intestine and caused outflow of fluid in a pulsatile manner at the slow wave frequency. The direction of propagation was always in an aboral direction. When the tissue was mounted in the opposite direction, the aboral end fixed to the pressure column and the oral end free to allow outflow, no outflow at the oral end occurred. In fact, the pressure dropped in the oral column identifying flow in aboral direction and a negative pressure gradient developed. The unidirectional propagation of slow waves is due to their intrinsic frequency gradient (Diamant and Bortoff, 1969; Huizinga et al., 1997). Hence, the correlation between propagating waves of transient intraluminal pressure (peristalsis) and slow waves can be easily demonstrated in segments of intestine in vitro. The above-described models of distention-induced peristalsis illustrate that two different mechanisms occur concurrently to induce aboral propagation of motor activity, one being a periodic wave of neural excitation that propagates aborally, the other, slow wave-driven peristalsis driving rhythmic motor activity in the same direction. These mechanisms may be aided by a neural reflex initiating ascending excitation oral to the distended segment (Tonini et al., 1996). The stomach has very regular periodic peristaltic waves of contraction when in the process of emptying. This peristaltic activity has underlying slow wave activity (Haba and Sarna, 1993; Van Nueten et al., 1990). Bauer provided evidence that these slow waves originated from the Auerbach’s plexus area (Bauer et al., 1985), which has a dense plexus of interstitial cells of Cajal (Christensen et al., 1992). In the guinea pig antrum, slow wave activity was recently shown in situ to be generated by interstitial cells of Cajal associated with Auerbach’s plexus (Dickens et al., 1999; Huang et al., 1999). These slow waves have the typical insensitivity to nifedipine and voltage changes. Interestingly, in circular muscle preparations without Auerbach’s plexus, slow wave-like activity is still recorded but this activity is abolished by hyperpolarization (induced by chromakalim) and not by nifedipine. The authors speculate that the slow wave activity might be related to the presence of intermuscular ICC. It is likely, however, that the rhythmicity of this activity resides in the smooth muscle cells themselves. Interesting evidence for the role of ICC as pacemaker activity has come from recent work from Hirst and coworkers (Dickens et al., 1999) and D.F. Van Helden (personal communication). In the antrum of the guinea pig, normal slow wave activity consisting of a voltage-independent pacemaker component and a voltage-dependent plateau is generated. An electrical pulse can generate a premature slow wave. In preparations without the myenteric plexus, slow wave activity does not occur but an electrical pulse generates the second component after a short delay. This provides interesting indirect evidence that the ICC produced the component that triggered the slow wave, and that the ICC-induced pacemaker activity can be replaced by an electrically induced depolarization. Motor Patterns Determined by Cholinergic Excitation in the Absence of Classic Slow Waves Mice with a spontaneous mutation in the Kit gene (WWv) produce an abnormal Kit protein that prevents 243 the normal development of ICC in the Auerbach’s plexus area of the small intestine and this musculature does not generate the classical slow wave. The small intestine upon receiving content from the stomach, does not exhibit regular rhythmic peristalsis but does generate some propulsive motor activity that, with great delay, moves content aborally (Der-Silaphet et al., 1998). What is the nature of this activity? Action potentials do not have a fixed frequency and can occur at entirely different frequencies at sites close to each other. How can electrical activity with such properties produce coordinated, propagating action potentials and, consequently, a propagating wave of increased intraluminal pressure? Although the WWv mouse, without marked stimulation of the musculature, does not show normal slow wave-driven peristalsis, significant distention in vitro has shown that regular rhythmic peristalsis can develop. The musculature, in the absence of classical slow waves, has the capability to generate slow wave-like activity that produces ‘‘slow wavedriven’’ peristalsis predominantly in the aboral direction (Fig. 4). In the W mutant mice, the frequency of the slow wave-like activity is remarkable constant at ⬃ 40 cpm, not different from that of normal slow wave activity! This oscillatory activity may originate in smooth muscle cells in response to neural stimulation, but it is also possible that ICC of the deep muscular plexus are involved (Hara et al., 1986; Jimenez et al., 1996). Cholinergic activity can cause general depolarization of a short segment of intestine that is so strong that action potentials are generated continuously, overruling the usual restrictions of slow wave activity if present. Such activity was noticed in the rat ileum, where a very strong contraction occurred in one part of the ileum and ‘‘moved’’ distally without immediate relaxation of the preceding segment. A motor pattern occurred that truly resembled the ‘‘squeezing of tooth paste,’’ in that an entire segment of intestine contracted for a few seconds. The driving force behind such a pattern of propulsive contractile activity is clearly the enteric nervous system. This motor activity occurred about 3 times per 10 minutes and nitrergic activity was shown to be part of the mechanism that controlled this frequency (Bercik et al., 1994). In the mid to distal mouse colon, slow wave activity is hardly ever observed (Bywater, personal communication). In this region, Bywater and colleagues (1989) noticed atropine-sensitive bursts of action potentials superimposed on slow, transient depolarizations of ⬃17 mV. These complexes occurred every 3 minutes and propagated aborally along the colon at a velocity of ⬃ 1 mm/s (Bywater et al., 1989; Lyster et al., 1995). The action potentials within a burst occurred associated with fast oscillations occurring at ⬃ 2/s. The periodicity of the bursts was determined, at least in part, by nitrergic enteric nerves responsible for electrical quiescence in between the bursts of activity. Interestingly, the periodic depolarizations were seen not to be sensitive to atropine, but due to periodic inhibition of otherwise continuous activity of nitrergic nerves (Spencer et al., 1998a,b). This periodic inhibition of nitric oxide release was accompanied by inhibition of neural activity (ATP release?) normally leading to inhibitory junction potentials. Bywater suggested that this motor 244 J.D. HUIZINGA Fig. 4. Distention induced periodic activity in the W/Wv mouse. A,B: Top three traces: electrical activity from 3 different sites 1 cm apart, recorded simultaneously. Bottom two traces: intraluminal pressure changes recorded simultaneously at intraluminal sites of electrodes 1 and 3. A: Without sufficient distention, action potentials occurred uncoordinated at the three sites. Pressure changes were also uncoordinated and no outflow occurred. B: Distention by 3.5 cm H2O of intraluminal pressure evoked periodic activity. The periods of activity of ⬃ 10 seconds duration occurred simultaneously at the different recording sites. Slow wave-like activity appeared on which action potentials were superimposed. The ‘‘slow waves’’ were related 1:1 to increases in intraluminal pressure and outflow pulses (j). Hence, the outflow was pulsatile in nature with each transient outflow associated with an individual propagating pressure wave of sufficient amplitude. The ‘‘slow waves’’ were different from classical slow waves in that they were blocked by L-type calcium blockers and hyperpolarization. Reproduced from Huizinga et al. (1998) with permission of the publisher. pattern represents the peristaltic activity of the mid to distal mouse colon (Bywater et al., 1998). Generation of propagating bursts of action potentials was also observed in the human colon, both in vitro and in vivo. In vitro, carbachol induces bursts of action potentials, often without any appreciable underlying depolarization (Huizinga and Waterfall, 1988). Interestingly, the development of such bursts is impaired in diverticular disease (Huizinga et al., 1999b). Normally, in vivo, bursts of action potentials (6–25-second duration) at 3 per minute or less, with very similar characteristics to those seen in vitro, are seen to propagate anally and produce flow of content upon muscarinic stimulation (Bueno et al., 1980; Schang et al., 1986). Reduced occurrence of such bursts is associated with constipation (Bueno et al., 1980). The periodicity of the motor complexes may reside in periodic presynaptic inhibition of nitric oxide release, which causes periodic depolarization. Since during this period cholinergic activity dominates, action potentials appear on top of the depolarizations, causing the periodic contractile activity. It is yet to be examined how such patterns of electrical activity relate to slow wave-like activity also generated by the human colon (Rae et al., 1998a). The guinea pig ileum is electrically quiescent without any stimulus applied to it, despite the presence of a network of interstitial cells of Cajal. All obvious morphological features of this ICC network are similar to those in the intestine of other species, such as the mouse (Christensen and Rick, 1987; Jessen and Thuneberg, 1991; Komuro and Zhou, 1996; Komuro et al., 1996; Zhou and Komuro, 1992). Electrical activity, however, does not show randomly propagating action potentials. Action potentials occur on transient depolarizations. Transient depolarizations occur in single isolated smooth muscle cells (Kohda et al., 1998) as well as in tissue evoked by cholinergic stimulation (Bolton, 1971). There appear to be several types of oscillatory activity occurring in the guinea pig (Huizinga et al., 1999c). In vitro, distention can be shown to produce a propagating contraction that empties the segment under investigation (Tonini et al., 1996) (Fig. 5). This contraction occurs rhythmically with sustained distention (Huizinga et al., 1999c) and cholinergically induces slow wave-like activity underlying the propagating contraction. The generation of this slow wave activity may involve interstitial cells of Cajal, but this point is still to be investigated. Hence, myogenic and neural activity work in concert to produce peristalsis. Neural activity includes ascending excitation as well as other more complex stretch sensitive neural circuitries (Kunze et al., 1998; Tonini et al., 1996). DIFFERENCES BETWEEN PATTERNS OF SEGMENTAL AND PROPULSIVE CONTRACTILE ACTIVITY In the small intestine, segmental and propulsive patterns of contractile activity can occur at irregular intervals. Both occur at the slow wave frequency and hence are likely to involve interstitial cells of Cajal. What are the factors determining the motor pattern when aborally propagating slow wave activity is likely underlying both phenomena? Once again, it is likely the MECHANISMS OF PERISTALSIS 245 Fig. 5. Distention-induced peristalsis in the guinea pig small intestine. The experimental setup as used by Costa and co-workers to initiate peristalsis and ascending excitation in a segment of intestine in vitro. Influx of fluid builds up distention of a segment of intestine (bottom trace). This distention will trigger a neural reflex initiating ascending excitation in a rhythmic fashion (top trace). At a certain threshold, a propagating wave of intraluminal pressure clears the content from the ‘‘anal’’ part of the intestine (middle trace). It can be seen that evoking ascending excitation is not enough to evoke peristalsis. A complex array of neural activities is triggering this peristalsis of which ascending excitation is only a part. This experimental setup is similar to that used with Figures 3 and 4 except that all recordings were made from the ‘‘anal’’ part and distention was maintained just above threshold, which results in rhythmic appearance of propagating contractions at the slow wave frequency. When this approach was taken with the guinea pig, cholinergically induced slow wave activity was associated with the peristaltic contractions (Huizinga et al., 1999c). Reproduced from Tonini et al. (1996) with permission of the publisher. pattern of nerve stimulation that determines the pattern of contraction. If a segment of intestine receives a more or less uniform level of excitation, all slow waves bear action potentials and the propagating nature of the slow waves sweeps the action potentials along the intestine and causes a propagating contraction. When the level of excitation is not uniform, action potentials will only occur in those regions where excitation produces sufficient depolarization for contraction generation. If such excitation were to occur over a significant period of time in one spot, contractile activity would be rhythmic at the slow wave frequency because only with every passing slow wave would action potentials be generated. It would not be propagating since the adjacent section would not be excited, hence no action potentials would be generated there. In this way a pattern of segmental contractile activity will be generated. An interesting new idea related to changing motor patterns comes from research from Thuneberg’s laboratory. Thuneberg discovered that distention results in a dramatic increase in special communication structures between smooth muscle cells and between smooth muscle cells and interstitial cells of Cajal (Thuneberg and Peters, 1999). Membrane extrusions from one cell form pegs that push into a neighbouring cell, forming a peg and socket junction. The quantity of such contacts can change from undetectable levels to hundreds of contacts per cell. Interestingly, the junctions are polarized with respect to ICC; the peg is always from smooth muscle cells, the socket always in ICC. Although this dramatic increase in peg and socket junctions occurs in experimental stretch, it still has to be discovered when they actually form in vivo. Furthermore, their physiological role has yet to be established. Thuneberg speculates that distention may induce peg and socket junctions to facilitate a motor pattern that is primarily segmental. Evidence for this is based on the observation that whenever tissue is fixed while engaged in segmental contractile activity, peg and socket contacts are abundant. Another possibility is that the junctions assist the role of ICC as stretch receptors. If stretch sensed by ICC is to be transmitted to the smooth muscle cells, existence of peg and socket junctions may greatly facilitate this. 246 J.D. HUIZINGA INTERSTITIAL CELLS OF CAJAL AND RHYTHMIC NEURAL EXCITATION OF GUT MUSCULATURE It has become clear from studies into distentioninduced peristalsis in the intestine and colon that the control of inhibitory innervation involving nitric oxide determines, to a great extent, the periodicity of rhythmic neural patterns of excitation (Der-Silaphet et al., 1998; Rae et al., 1998b; Spencer et al., 1998a). Since evidence is emerging that ICC are involved in inhibitory innervation to the gut musculature (Sanders, 1996; Ward et al., 1998), it will be interesting to study the specific role of ICC in rhythmic patterns of neural excitation. A well-studied motor pattern involving rhythmic activity of the enteric nervous system is the ‘‘migrating myo-electric complex’’ or MMC, which occurs in the empty stomach and intestine. The initiation of the MMC resides in the enteric nervous system and in part the CNS through vagal pathways (Hall et al., 1982, 1984; Rudge et al., 1990; Sarna et al., 1984; Szurszewski, 1969). In humans, every 1.5 hours a section of stomach or intestine receives intense stimulation from cholinergic motor neurons that derive from the vagus or the enteric nervous system. Two propagating activities are superimposed. First, the MMC, as a band of neural excitation, travels slowly from the LES towards the distal intestine. Secondly, within the segment of stomach or intestine under activation, rhythmic slow wave driven peristalsis plays an important role in moving contents anally (Hall et al., 1982). It would be interesting to study the specific interactions between those nerves involved in initiating the MCC and interstitial cells of Cajal. Interestingly, the MMC is one of the peristaltic motor patterns not requiring distention for its initiation. The existence of multiple control levels for gastrointestinal motility has always been recognized. In 1899, Bayliss and Starling noticed myogenic waves of contractions with associated transit of content, in addition to their discovery of the law of the intestine (Bayliss and Starling, 1899). The characteristics of myogenic control have been further elucidated by Alvarez (Alvarez, 1948) and, subsequently, by many others notably Diamant and Bortoff (1969) and Szurszewski et al. (1970). A synthesis of myogenic and neural control mechanisms was given by Costa and Furness (1982) and Weisbrodt (1987). The present review incorporates the interstitial cells of Cajal into the control mechanisms of peristalsis. No significant motor activity occurs without excitation of the musculature by enteric nerves. Once activated, however, the gut musculature exhibits its intrinsic control mechanisms. Interstitial cells of Cajal generate slow wave activity passed on to involve the whole musculature. This slow wave activity permits action potentials to occur in a rhythmic and propagating manner, and such slow wave-driven peristaltic activity is central to many motor patterns of peristalsis. ACKNOWLEDGMENTS The Medical Research Council of Canada is acknowledged for supporting most of the research from my laboratory mentioned in this review. Yaohui Zhu recorded the spontaneous inward currents from an ICC shown in Figure 2. REFERENCES Alvarez W. C. 1948. 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