A new model of pacing in the mouse intestine

E. E. Daniel, Geoffrey Boddy, Alicia Bong, and WooJung Cho

Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, T6G 2H7 Canada

Submitted 12 May 2003 ; accepted in final form 1 October 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A simple model of pacing in mouse intestine to longitudinal (LM) as well as circular muscle (CM) has been developed. Undissected segments of LM or CM from mouse ileum or jejunum were prepared to record contractions, nerve functions were inhibited, and regular spontaneous contractions were recorded. These had the properties expected of interstitial cells of Cajal (ICC) paced contractions: ileum slower than jejunum, inhibited but not abolished by nicardipine, reduced in frequency by cyclopiazonic acid, abolished by Ca2+-free media, and high temperature dependence (Q10~2.6-3.2). Nicardipine significantly reduced the pacing frequency in LM and CM. Intestinal segments from W/WV mice had few irregular contractions in CM but had regular contractions in LM. Other differences were found between LM and CM that suggest that the control of pacing of LM differed from pacing of CM. Moreover, both LM and CM segments in wild-type and W/WV and after cyclopiazonic acid responded to electrical pacing (50 V/cm, 50 or 100 ms) at 1 pulse per second. Temperature <26°C inhibited electrically paced contractions in CM. These findings suggest that the current models of ICC pacing need to be modified to apply to intact segments of mouse intestine.

cyclopiazonic acid; interstitial cells of Cajal


THE CURRENT UNDERSTANDING of pacing in the small intestine depends on studies of slow waves in mouse tissue that have been extensively dissected and on currents and other properties of interstitial cells of Cajal (ICC) that have been isolated and cultured from newborn mice (15-17, 19, 22, 25, 26, 30-34, 36-38, 41, 42, 44, 46, 49, 52-55, 56, 58). Furthermore, only one report of possible pacing of mouse intestine longitudinal muscle (LM) could be found in the literature (58). In other species, LM and circular muscle (CM) have slow waves believed to be paced simultaneously the by ICC of the myenteric plexus (1-3, 6, 7, 14, 18). In mutant mice, which lack an ICC network in the myenteric plexus, slow waves are missing from the dissected CM even though the ICC network of the deep muscular plexus appears to be intact in fixed specimens. (26, 30, 37, 38, 41, 42, 45, 52). In canine intestine (18, 27), slow waves can also be driven by a secondary network of ICC in the deep muscular plexus. In the mouse intestine, the integrity of this network after dissection and recording from CM has never been established.

When ICC-paced slow waves in mouse intestine reach the threshold for opening L-type Ca2+ channels, Ca2+ currents and action potentials are produced that lead to contractions (6, 16, 22, 25, 34, 37, 38, 42). Also, slow waves can be considered action potentials because they can induce contractions in the absence of spike-type action potentials. Therefore, ICC-driven contractions should be reduced but not necessarily abolished after block of L-type Ca2+ channels. Furthermore, ICC currents are reported to be susceptible to block of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps by cyclopiazonic acid (CPA), so that slow-wave frequencies are decreased (35, 36, 55). Thus ICC-driven contractions should also be decreased in frequency by CPA. Finally, the CM of intestine of W/WV mice, which lack ICC in the myenteric plexus, should lack regular spontaneous contractions. The status of ICC-driven contractions in the LM of intestine in these mice is unknown.

In intestine of mouse myenteric plexus and other species, ICC are interconnected by visible gap junctions (8, 9, 36, 45, 47, 48, 53), and ICC-driven slow waves and contractions are assumed to be transmitted to muscle through gap junctions that provide low-resistance conduits for current flow (8, 9, 18, 22, 41, 42, 53), and the muscle is assumed to follow passively. However, structural gap junctions have never been found connecting ICC of the myenteric plexus with either the LM or CM of mouse intestine (9, 38, 47, 48, 53). In the canine intestine, only rare gap junctions have been found connecting the network of ICC in the myenteric plexus to CM, and no such connection has been found to LM (9, 10). Furthermore, in canine intestine and colon (8) and in mouse intestine (43), we have observed that gap junction uncouplers do not inhibit transmission of slow waves or contractions to muscle. Thus how the ICC pacing is transmitted to muscle is unclear.

We showed in canine intestine that slow waves could be paced by electrical pulses of 50-100 ms provided the ICC network of the myenteric plexus was intact (5). Presumably, these pulses initiated pacing currents in the myenteric plexus ICC network, and smooth muscle cells were responding to the them. If the myenteric plexus ICC network of mouse intestine is required for electrical pacing, then electrical pacing of CM should be absent in W/WV mice and reduced in frequency by CPA. On the other hand, if electrical pacing is effective because the muscle and/or intramuscular ICC (ICC-IM) are coupled and receive electrical signals from either ICC or an external current source, as in guinea pig antrum (12, 13, 20, 21, 40), the responses to electrical pacing will reflect the status of the response system (ICC-IM plus muscle) and not the myenteric plexus ICC.

Our objective was to develop a simple in vitro system to evaluate ICC pacing in the mouse intestine without dissection of the muscle layers and with the ability to evaluate pacing in LM as well as CM. We tested our system by comparing the responses of our model with those known for slow waves of the isolated CM of the mouse intestine. Our results suggest that our model reflects ICC-driven pacing but that the current understanding of the controls of pacing may be incomplete.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of tissue. Male, 6- to 12-wk-old BALB/c mice or W/WV mice (from Jackson Laboratories) were killed by cervical dislocation following the guidelines of our institutional Animal Ethics Committee. After the opening of the abdominal wall, the digestive tract, beginning from the level of the stomach to the rectum, was removed from the mouse. The tissue was immediately placed into a beaker of Krebs-Ringer solution that had been bubbled at room temperature (21-22°C) with carbogen (95% O2-5% CO2) for ~5 min before the dissection. In a petri dish filled with Krebs solution continuously bubbled with carbogen, ileal (considered to be the distal third of the intestine) or jejunum (considered to be half of the intestine and located proximal to the ileum) tissue was isolated and cut into ~0.5 (for CM)- or 1- to 1.5-cm (for LM) segments in preparation to mount onto glass holders with electrodes.

To study LM contractions, a tissue segment was placed between two platinum concentric circular electrodes and tied to a hook at the bottom of the electrode holder with silk suture thread. The top of the tissue was also tied with thread and attached to a strain gauge. To study CM contractions, one side of a thin metal triangle was slid through the lumen of the tissue segment. The triangle was then hooked together. A stainless steel rod attached to the bottom of the electrode holder was inserted into the lumen of the tissue under the metal triangle. Suture thread, attached at the apex of the triangle opposite to the tissue, was tied to the strain gauge. Two thin platinum rods, situated parallel to and on either side of the tissue, were used to stimulate the tissue electrically.

Two longitudinal and two circular preparations were mounted and placed into muscle baths, filled with 10 ml Krebs solution, and bubbled continuously throughout the experiment with 95% O2 and 5% CO2 and maintained at a temperature of 37°C. The thread connected to each tissue segment was then tied to a string gauge (Grass FT-03). The tissues were subjected to slight tension, just sufficient to remove slack in the thread, and allowed to stabilize for 10 min. Tissue contractile activities were recorded on a Beckman Dynograph 611.

Experimental protocol. The tissues were electrically field stimulated (EFS) to test nerve activity [parameters: 40 V/cm, 0.5 ms, and 5 pulses per second (pps)]. LM always responded with contraction, whereas CM usually responded with relaxation. TTX (10-6 M) and NG-nitro-L-arginine (L-NNA, 10-4 M) were added to the baths to eliminate enteric nerve function and nitric oxide (NO) production. In some experiments, conotoxin GVIA at 10-7 M was also added, but it did not alter subsequent responses and was usually omitted. Within 1 min, CM contraction amplitudes increased. After 5 min, nerve activity was tested again by EFS. If there was any response in the tissue, TTX was added again until all enteric nerve function was blocked. The frequencies of slow wave-driven contractions were measured before and after TTX and L-NNA. After nerve activity was blocked by TTX and L-NNA, the experiment was carried out using the pharmacological agents under investigation. In some experiments, EFS was carried out at 50 V/cm, 50- or 100-ms pulses, and 60 Hz to pace segments electrically. At the end of each experiment, all tissues were washed twice with 10 ml Ca2+-free Krebs to relax tissues to basal passive tension and abolish spontaneous contractions.

Experimental procedures. Several types of procedures were used, and they are described in depth in RESULTS. In general, for each experiment, one LM and one CM segment were used as experimental tissue and another of each as a time control. Frequency and amplitude measurements were made every 5 min. Frequencies were measured over a period of at least 20 s. Contractions were regular and stable in time controls over time (see RESULTS). Amplitudes of contractions were determined as the values above the passive tension determined at the end of the experiment. They were measured by evaluating individual contractions over at least nine contractions and calculating a mean and standard error. In many cases, only the data before and after final measurements for each experimental intervention are presented. In the case of contraction amplitudes, the results were normalized to the mean values in the control periods, set as 100%.

For study of temperature effects, contractions of segments were recorded at room temperature and then after warming to 38°C, followed by recordings as the temperature fell after the heater was turned off. The n values represent the number of mice whose intestine provided segments for study.

Analysis of data. Measurements were entered into GraphPad Instat and analyzed by appropriate methods as explained in the figure legends. Figures were plotted in Prism 3. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 designate probabilities.

Drugs and solutions. TTX and T conotoxin GVIA were from Calbiochem-Novabiochem (San Diego, CA) or from Alomone Laboratories (Jerusalem, Israel). L-NNA, CPA, and nicardipine were from Sigma-Aldrich (Oakville, Canada). As CPA was dissolved in DMSO, the solvent was added to time control tissues in equivalent amounts. The final concentration of DMSO did not exceed 0.1% and had no significant effects on control tissues. DMSO was from Sigma-Aldrich. Krebs solution contained (in mM) 115.5 NaCl, 21.9 NaHCO3, 11.1 glucose, 4.6 KCl, 1.16 MgSO4·H2O, 1.16 NaH2PO4·H2O, and 2.5 CaCl2·H2O. Ca2+-free Krebs solution contained (in mM) 115.5 NaCl, 21.9 NaHCO3, 11.1 glucose, 4.6 KCl, 1.16 MgSO4·H2O, 1.16 NaH2PO4·H2O, and 1.0 EGTA.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effects of TTX and L-NNA. Figure 1 shows representative effects of EFS at 50 V/cm, 0.5-ms pulses at 5 pps for 6-8 s. In all cases, over 200 to date, LM contracted. CM relaxed with an excitatory "off" response following inhibition of contractions in most cases. Five minutes after 10-6 M TTX and 10-4 M L-NNA, EFS had no effects. After TTX and L-NNA, frequency of LM did not change significantly (0.074 ± 0.492, n = 209, paired comparison), whereas that of CM increased significantly (3.915 ± 0.776, n = 130; P < 0.0001, paired comparison). Amplitudes of CM contractions were also significantly increased, whereas those of LM were slightly reduced (not shown). Subsequent stimulation with 100-ms pulses at 1 pps produced contractions at these frequencies.



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Fig. 1. Representative tracing of responses of longitudinal (LM; A) and circular muscle (CM; B) to electrical field stimulation (EFS) at 50 V/cm, 5 pulses per second (pps), and 0.5-ms pulses (portion indicated by bar). In all cases, LM segments responded by increased amplitude of spontaneous contractions, and >90% of CM segments responded with inhibition followed by a rebound excitation or, if there were no spontaneous contractions, by a following rebound excitation. Five minutes after 10-6 M TTX and 10-4 M NG-nitro-L-arginine (L-NNA), there were no responses to EFS at the above parameters, but the tissues contracted when pulses of 100 ms at 1 pps were applied (see Fig. 8).

 



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Fig. 8. Excerpts from traces from experiments illustrating responses from BALB/c (A) and W/WV (B) mouse intestinal segments at various stages of our experimental protocols, before and after TTX and L-NNA, after subsequent Nicard, and then after CPA. Finally, the responses to 1 pps stimulation at the end of the experiments are shown. Zero tone, except in the case of responses to 1-pps stimulation, is marked on each segment. Where amplification changes had to be made during the experiments, they are shown on the excerpt.

 
Comparison of ileum and jejunum after TTX and L-NNA. After block of nerve function, the contraction frequencies of both LM and CM segments were greater in jejunum than in ileum. For LM, mean jejunal frequency was 56.60 ± 0.98 (n = 80) compared with an ileal frequency of 49.48 ± 0.98 (n = 30; P < 0.0001) using unpaired comparisons. For CM, mean jejunal frequency was 50.23 ± 0.76 (n = 74) compared with an ileal frequency of 44.61 ± 1.12 (n = 31; P < 0.0001) using unpaired comparisons. Amplitudes were not significantly different (not shown).

Comparison of LM to CM in the same region. Analysis of these same data showed that the frequencies of contraction were significantly greater (P < 0.0001) in LM compared with CM in both jejunum and ileum. Because higher frequencies may occur at proximal sites, we always chose adjacent segments of LM and CM with the CM from the proximal site. Figure 2 shows representative examples. Contraction amplitudes were not compared, because the comparison of results from the two different tissues is inappropriate.



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Fig. 2. A: as shown here in representative experiments, segments of LM had slightly higher frequencies than adjacent proximal segments of CM. Also, nicardipine (10-6 M) decreased the frequencies and amplitudes of contraction in all segments of both LM and CM. Figure 2B shows similarly representative experiments in which cyclopiazonic acid (CPA) reduced frequencies of contractions in both LM and CM but increased amplitudes of contractions in LM and had no effect in CM.

 

Effects of nicardipine. Nicardipine (1 µM) decreased the frequencies of contraction of LM and CM intestinal segments slightly, but within 10 min, it was more significant (Figs. 2 and 3A). It rapidly decreased the amplitudes of contraction to about one-half control values by 10 min in both LM and CM (Figs. 2 and 3B).



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Fig. 3. Effects of nicardipine (Nicard; 1 µM) on frequencies and amplitudes of contraction are shown on frequencies of LM (A) and CM (B) and amplitudes of LM (C) and CM (D). Analyses were by paired comparisons before and after Nicard. There were no changes in frequencies or amplitudes of time controls (not shown).

 

Effect of CPA. At 10 µM, CPA significantly decreased the frequencies of ICC-driven contractions by at least one-half within 20 min (Figs. 2 and 4, A and B). Effects on frequency were apparent within 1-2 min and became stable after ~15 min. Within 1-2 min, tone increased in LM but not much in CM. This accounted for most of the increase in tone. Only very rarely did contractions cease completely and always in CM, not LM. In LM, CPA significantly increased contraction amplitudes, measured from passive tension after exposure to Ca2+-free Krebs, which also abolished all phasic contractions. It had no effect on contraction amplitudes in CM (Figs. 2 and 4, C and D).



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Fig. 4. A and B: effects of 10 µM CPA on the frequencies of contraction as %initial frequencies. These were decreased by about one-half in LM (A) and to 40% in CM (B) but not by the DMSO used to dissolve CPA (n = 9 or 10 in all cases). Paired comparisons were made. C and D: in the same segments as used in Fig. 7, the amplitudes of contraction, as %initial amplitudes, were unchanged in CM (D) but increased ~3-fold in LM (C). Paired comparisons were made.

 



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Fig. 7. Effects of 10 µM CPA on tone (as distinct from total contraction amplitude including phasic contractions) following 10-6 M Nicard or a time control in the 11 LM and CM segments analyzed in Figs. 5 and 6. A: results for LM. B: results for CM. Tone values are normalized to initial tone. The results show that CPA still produced a tone increase in LM and CM after Nicard. One-way ANOVA with Tukey-Kramer postcomparison tests.

 
Effects of CPA after nicardipine. In a separate series of experiments, 1 µM nicardipine was given to one of two segments of LM or CM 10 min before 10 µM CPA. Figure 5, A and B, shows the effects on frequencies, and Fig. 6, A and B, shows the effects on amplitudes. Prior nicardipine did not prevent or markedly alter the decrease in frequency by CPA. It did, however, reduce markedly the increase in amplitude in LM by CPA. Figure 7 shows the changes in tone, excluding phasic contractions and comparing with initial tone in these experiments. Nicardipine decreased initial tone in LM but not CM, which had very little to begin with. It did not prevent an increase in tone by CPA in either LM or even in CM.



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Fig. 5. Effects of 10 µM CPA on contraction frequencies following 10-6 M Nicard or a time control in 11 LM and CM segments. A: in LM, Nicard decreased contraction frequencies and CPA decreased it further, and effects were approximately additive. Note that there are 2 time controls in tissues treated with CPA alone because there were 2 periods before addition of Nicard. B: in CM, Nicard decreased frequencies slightly but insignificantly, and CPA had effects similar to those in time controls without Nicard. One-way ANOVA with Tukey-Kramer postcomparison tests.

 


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Fig. 6. Effects of 10 µM CPA on contraction amplitudes following 10-6 M Nicard or a time control in the same 11 LM or CM segments as in Fig. 5. For LM (A) and CM segments (B), prior Nicard decreased total contraction amplitudes and largely prevented the CPA-induced increase in contraction amplitude. One-way ANOVA with Tukey-Kramer postcomparison tests. See Fig. 7.

 

Responses to EFS of 100 ms and 1 pps after nicardipine and CPA. All segments contracted initially and after nicardipine and CPA to these pulses. Figure 8 shows typical responses of LM and CM after TTX and L-NNA and after nicardipine and CPA in the same segments.

Responses at different temperatures. Frequencies were ~15 per minute at 22°C and 45-60 per minute at 38°C. Figure 9, A and B, shows graphs of the effects of temperature on frequencies in ileum and Fig. 9, C and D, provides data for the jejunum. The slopes of these graphs yielded Q10 values of 2.6 for LM and CM of the ileum and 3.2 for LM and CM of the jejunum from linear regression. Note that the differences in frequencies between the jejunum and the ileum and between LM and CM were maintained.



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Fig. 9. The effects of temperature on the frequencies of ileal contractions are shown in A (LM) and B (CM). Those in jejunum are shown in C (LM) and D (CM). Frequencies were linearly related to temperature, with slopes of 2.6 for both LM and CM in ileum (linear regression analysis, r2 = 0.909 in both cases). For CM, there was a slight significant deviation from linearity at lower temperatures. The effects of temperature on the frequencies of jejunal contractions were also linearly related to temperature, with slopes of 3.3 for LM and 3.2 for CM (linear regression analysis, r2 = 0.845 for LM and 0.934 for CM). There was no significant deviation from linearity.

 

Contraction in LM and CM of W/WV mouse intestine. In seven W/WV mice, contractile activities of LM and CM segments from the ileum (16) and jejunum (8) were studied. Many of these had neural responses to EFS-like controls: 15 of 16 ileal segments and 10 of 10 jejunal segments of LM were contracted, whereas 11 of 16 ileal and 5 of 10 jejunal CM segments were inhibited by EFS. After TTX and L-NNA, regular phasic activity was present in 13 of 15 ileal and 7 of 9 jejunal LM segments. In CM, 13 of 16 ileal and 7 of 9 jejunal segments had phasic activity, intermittent in 10 ileal and 7 jejunal segments. Figure 8 shows representative traces.

Effects of nicardipine. After nicardipine, 12 of 15 ileal LM segments and 7 of 9 jejunal LM segments had regular phasic activity, reduced in amplitude but not frequency (Fig. 10A). Thus nicardipine abolished phasic activity in very few LM segments. Eight of thirteen ileal CM segments had phasic activity, with four of them intermittent, after nicardipine (Fig. 10B). Thus nicardipine abolished phasic activity in 5 more of the 13 CM segments of ileum. Only four of nine jejunal CM segments had phasic activity, with two of them intermittent after nicardipine. Thus nicardipine abolished phasic activity in most CM segments from both regions.



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Fig. 10. Frequencies of contractions of LM and CM ileal segments from W/WV mice are shown in A and B, respectively. Only segments that had phasic contractions though the entire protocol were included. Note that neither Nicard nor CPA affected contraction frequency of either LM or CM segments. Frequencies of contractions of LM and CM jejunal segments from W/WV mice are shown in C and D, respectively. Only segments that had phasic contractions though the entire protocol were included. TTX increased frequencies of CM segments as in control mice, but neither Nicard nor CPA affected contraction frequency of either LM or CM segments.

 

Effects of CPA. In 12 ileal LM segments, CPA increased the frequency of phasic activity during the first 5 min while tone increased (Fig. 10A). Later, frequency stayed increased in six segments or remained unchanged in five. By 20 min, it was reduced in three and abolished in one segment. Overall, there were no significant changes in frequency after 20 min for the 11 segments from which complete data were obtained (Fig. 10A). In seven jejunal LM segments, CPA increased frequencies in most segments initially during the period of initial tone increase. After 20 min, it reduced frequencies in two segments and abolished phasic activity in one other. Overall, there were no significant changes in frequency at that time in six segments from which complete data were obtained (Fig. 10C). In eight ileal CM segments for which complete data were obtained, no significant changes in frequency were found (Fig. 10B). The frequencies of CM contractions were notably slower than in LM of ileal segments, likely because several CM segments were contracting only intermittently. In the three jejunal segments for which complete data were obtained, there was a significant increase in frequency after TTX and L-NNA, but no other significant changes occurred (Fig. 10D).

Amplitudes of W/WV intestinal segments. Nicardipine decreased the amplitudes of contraction in ileal LM and CM, the latter not significantly. However, although 10 µM CPA restored contraction amplitudes, there was no increase over control levels after nicardipine or in the time controls, and changes were not significant (Fig. 11, A and B). There was the usual increase in tone by CPA in LM, however, by 63% (P < 0.01) compared with control and by 115% compared with tone after nicardipine (P < 0.001). In the time controls, CPA did not significantly increase tone. The failure of total contraction amplitudes to increase was the result of a marked decrease in the amplitudes of phasic activities. In jejunum, insufficient numbers of segments with regular phasic activity after TTX and L-NNA prevented running time controls. Nicardipine reduced contraction amplitudes in LM, and CPA restored the amplitudes, but without significant change from amplitudes after nicardipine or controls (Fig. 11C). In CM of jejunum, there were only six segments that had phasic activity after nicardipine, and only four segments had activity after CPA. The only significant changes were increases after CPA (Fig. 11D).



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Fig. 11. Amplitudes of contractions after CPA or Nicard followed by CPA are compared with initial values of ileal segments of LM and CM from W/WV mice in A and B, respectively. All tissues that had phasic contractions throughout the experiment were included, as were some time controls. Nicard reduced contraction amplitudes significantly in the case of LM, but there were no other significant changes. Similarly, C and D show amplitudes of contractions compared with initial values in jejunal segments of LM and CM, respectively, from W/WV mice. All tissues that had phasic contractions throughout the experiment were included, but there were too few time controls for inclusion. Nicard reduced contractions amplitudes in the case of LM, CPA increased it (in CM) or restored it (in LM).

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Our main findings are that isolated, undissected segments of LM and CM mouse intestine behave as if contractions are paced by ICC after nerve functions are abolished by TTX and L-NNA. Thus contractions were regular, highly temperature dependent, faster in jejunum than ileum, reduced markedly in amplitude by block of L-type Ca2+ channels, and reduced markedly in frequency by inhibition of SERCA pumps by CPA. CM contractions before nerve block were under the influence of NO-mediated inhibition, and they were increased by TTX plus L-NNA in both frequency and amplitude. LM contractions were under the influence of excitatory nerves, but block of nerve activity did not affect frequency and may have reduced amplitudes.

One unexpected finding was that LM contractions were significantly more frequent than those of CM from adjacent regions. This is unlikely to occur in vivo if both layers are paced by the same ICC network, and recent evidence suggests that LM and CM in mouse segments contract simultaneously (39). The reasons for our finding are unclear but may relate to the sizes or locations of the different preparations used. We routinely ensured that the CM segment was from a site adjacent to but orad from the LM segment. Thus a higher intrinsic frequency from an orad location of LM segments is not an explanation of the higher frequencies of LM compared with CM segments. We tested whether different applied tensions could explain the difference, but deliberate changes in applied tension did not affect the frequencies of LM segments. There were other unexpected findings. Block of L-type Ca2+ channels also reduced frequencies of contractions slightly but significantly. Furthermore, it only occasionally abolished ICC-paced CM contractions. All segments responded to long-duration (100 ms) pulses from EFS at 1 pps before and after nicardipine and CPA. This implies that all were capable of contractions at higher frequencies than their intrinsic frequencies.

As expected if ICC are absent, CM segments from W/WV mice often lacked regular phasic activity. It was usually absent or intermittent after nicardipine (only 4 of 16 ileal CM segments and 2 of 10 jejunal CM segments had persistent, nonintermittent phasic activity after TTX, L-NNA, and nicardipine). Enteric nerve responses in these tissues resembled those of BALB/c mouse intestines, excitation of LM, and inhibition of CM segments. However, LM segments of both ileal and jejunal segments were clearly capable of phasic activity after TTX, L-NNA, and nicardipine. They also responded differently to CPA, frequently showing increased frequency initially and no change in frequency after 20 min. All tissues responded to EFS of 100 ms and 1 pps. These results suggest first that in the normal mouse intestine, nonneural control of CM and LM segments occurs similarly, with reduction of ICC-driven contraction amplitudes by L-type Ca2+ channel block and reduction in frequency by lowered temperature or inhibition of the SERCA pump.

These are criteria, described in the introduction, which distinguish ICC-paced from other neural or other contractile activities. These results also make clear that the reported absence of myenteric plexus ICC in W/WV mouse intestine does not completely abolish contractions that meet the criteria that they occur after both nerve function and L-type Ca2+ channels are blocked. In the case of CM, such contractions are uncommon and may relate to the occasional presence of ICC cells in the myenteric plexus, observed by electron microscopy (37) and confirmed in our tissues by using immunocytochemistry (unpublished data).

In the case of LM, the absence of the normal network of ICC in the myenteric plexus does not abolish regular pacing activities, because most segments still possessed regular contractions after TTX, L-NNA, and nicardipine. Possibly, the absence of these ICCs uncoupled slow waves from their control, because CPA no longer decreased contraction frequencies. We have found no reports in the literature about slow waves in LM of mouse intestine, but they are present in LM of most mammalian species, presumably driven by ICC (1-3, 6, 7, 14, 18). It is clear also that control of intestinal motility in the mouse intestine depends on interactions between neural intestinal reflexes, in which LM as well as CM play roles, and on ICC-paced contractions (11, 23, 24). Thus control of LM contractile activity by myenteric plexus ICC is likely essential to normal motility. A major premise of ICC control over intestinal contractile activity is that the muscle responds passively to the currents supplied by the ICC and transmitted to the muscle through gap junctions (8). This is based on the absence of slow waves in tissue separated from ICC networks in various species (18, 26, 27, 53, 58) and on the evidence for decremental spread from ICC networks in canine colon (41). However, we previously showed that inhibitors of gap junction conductance do not block ICC-driven contractions in the canine intestine or colon (8) and in mouse intestine (43). In the canine intestine, we found that slow waves occurred in response to long-duration EFS pulses only if the ICC network of the myenteric plexus was present (5), although the network of the deep muscular plexus can drive slow waves in the absence of the myenteric plexus (28).

Thus we expected the responses of the mouse intestine to long-duration EFS to depend on the presence of and functioning of the myenteric plexus. However, responses were present at 1 pps after CPA had reduced the frequencies of ICC-driven contractions by half in normal intestine as well as in intestine from W/WV mice. They were also present after CPA reduced frequencies by half in normal mouse intestine. This suggests that the muscle does not depend on ICC activity to respond to depolarizing pulses. Whether this is a result of the excitability of the muscle layers or of ICC in the deep muscular plexus is unclear. In the mouse gastrointestinal tract, ICCs in the muscle layer have been shown to be sites of nerve modulation (41, 50, 51, 55, 57). It is assumed that they transmit the neural message through gap junctions to smooth muscle. These ICC-IM also play roles in pacing as has been shown recently in guinea pig and mouse antrum or pylorus.

ICC-IM can amplify the depolarizing message from ICC (12, 13, 20, 21, 40). We (unpublished data) and others (Ref. 48; also H. Mikklesen, personal communication) have failed to find ICC-IM in the mouse intestine. However, in mouse intestine, the ICC network of the deep muscular plexus may function as ICC-IM and may be involved in responses to depolarizing stimuli.

In conclusion, this study shows that the control of pacing in the mouse intestine can be studied without extensive dissection and pinning of muscle, that the LM is usually controlled by similar mechanisms to CM, but that in intestine from W/WV mice, there is a robust pacing system in LM that does not require the presence of the network of ICC in the myenteric plexus. In the absence of that network, pacing is no longer dependent on the function of SERCA pumps.


    ACKNOWLEDGMENTS
 
We acknowledge the contributions of T. Schultz to completing studies with CPA.

GRANTS

The Canadian Institutes of Health Research supported these studies.


    FOOTNOTES
 

Address for reprint requests and other correspondence: E. E. Daniel, Rm. 9-10, Medical Sciences Bldg., Dept. Of Pharmacology, Univ. of Alberta, Edmonton, AB, T6G 2H7 Canada (E-mail: edaniel{at}ualberta.ca).

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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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