Faculty of Medicine and Dentistry, Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada
Submitted 27 July 2004 ; accepted in final form 7 October 2004
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ABSTRACT |
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interstitial cells of Cajal; gradient of pacing frequency; nicardipine
However, this theory is not supported by other studies (5, 6, 30) that have found an absence of gap junctions connecting ICC-MP to muscles and the absence of inhibition of pacing after gap junction uncouplers using small segments of LM or CM. Furthermore, the role of Ca2+ release from ICC ER has been questioned by evidence that putative blocking of Ins(1,4,5)P3-mediated release from ER, such as by 2-aminoethoxyphenyl borate is likely nonspecific, whereas other agents, such as xestospongin C, are ineffective (1). Also, the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump blockers cyclopiazonic acid or thapsigargin have not completely blocked pacing in intact muscle segments, as reported in cultured and dissected intestine (1). Inhibition of mitochondrial Ca2+ uptake by carbonyl cyanide-p-(triflouromethoxy)phenylhydrazone also was questioned, because of its serious effects on cellular function.
Studies of segments of intestine from W/WV mice revealed that the LM segments had a robust pacing function not abolished by block of L-Ca2+ channels or affected by depletion of ER Ca2+ stores (4). There appear to be no records in the literature of intracellular recording from intestinal LM cells in wild-type or W/WV mice. These findings suggest that models of pacing in mouse intestine by ICC-MP, based on studies of highly reduced systems, need to be reevaluated by using more intact systems.
The aims of this study were to evaluate the role of L-Ca2+ channels in pacing in segments of wild-type and W/WV mice, and to compare the mechanisms of pacing in LM segments of W/WV mice with those of LM and CM segments from wild-type mice.
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MATERIALS AND METHODS |
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Male, 6- to 12-wk-old Balb/C, W/W++, or W/WV mice (from Jackson Laboratories, Bar Harbor, ME) 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. Tissue was immediately placed into a beaker of Krebs Ringer solution that had been bubbled at room temperature (2122°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 11.5 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, the open 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 to secure the triangle and provide slight tension. 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 LM and two CM preparations were mounted and placed in muscle baths, filled with 10 ml Krebs solution and bubbled continuously throughout the experiment with 95% O2-5% CO2 and maintained at a temperature of 37°C. The thread connected to each tissue segment was then tied to a strain gauge (Grass FT-03). 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
Tissues were electrically field stimulated to test nerve activity (parameters: 50 V/cm, 0.5 ms, and 5 pps). LM from wild-type mouse intestines always responded with contraction, whereas CM usually responded with relaxation. TTX (106 M) and N-nitro-L-arginine (L-NNA; 104 M) were added to the baths to eliminate enteric nerve function and NO production. Within 1 min, CM contraction amplitudes increased. After 5 min, nerve activity was tested again by electrical field stimulation. If there was any response in the tissue, TTX was added again until all enteric nerve function was blocked. 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, tension was adjusted to provide maximum spontaneous contractions and the experiment was carried out by using the pharmacological agents under investigation. In some experiments, electrical field stimulation was carried out at 50 V/cm, 10- or 30-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 with 1 mM EGTA to relax tissues to basal passive tension and abolish spontaneous contractions.
Experimental Procedures
Several types of procedures were used, and they will be described in depth in RESULTS. In general, for each experiment, one LM and one CM segment was used as experimental tissue and another of each as time control. Frequency and amplitude measurements were made every 510 min. Frequencies were measured over a period of at least 20 s. Contractions were regular and stable in time control over time (see RESULTS). Amplitudes of contractions were determined as the values above the passive tension determined at the end of the experiment.
Amplitude was measured by evaluating individual contractions over at least nine contractions and calculating a mean ± SE. In many cases, only the data before each experimental intervention and final measurements are presented. In the case of contraction amplitudes, results were normalized to the mean values in the control periods set as 100%. The n values represent the number of mice whose intestine provided segments for study.
Analysis of Data
Measurements were entered into Graph Pad Instat and analyzed by paired comparison, using Student's paired t-test or ANOVA as appropriate. Figures 17 were plotted in Prism 3. Statistical significance was *P 0.05, **P
0.01, ***P
0.001, and ****P
0.0001. Comparisons between experimental tissues and time controls are #P
0.05, ##P
0.01, ###P
0.001, and ####P
0.0001.
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Pharmacological agents. TTX was from Alomone Laboratories (Jerusalem, Israel); L-NNA, BAY K 8644, and nicardipine were from Sigma-Aldrich (St. Louis, MO). Because some drugs were 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.
Solutions. Krebs solution was (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 was (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.
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RESULTS |
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Nicardipine is a dihydropyridine, L-Ca2+ channel antagonist that binds with highest affinity to the depolarized, inactivated state of the channel, causing the channel to remain inactivated (11, 33). It has the advantage over some other L-Ca2+ channel antagonists in that it is not light sensitive. We have previously demonstrated the effect of 1 µM nicardipine on intestinal smooth muscle pacing to reduce the frequency and amplitude in both LM and CM. In this study, we examined the effect of 0.3, 1, and 3 µM nicardipine added consecutively and cumulatively to LM and CM strips from ileum and jejunum independently (Figs. 1 and 2). Final measurements of frequency and amplitude were taken 15 min after application of nicardipine at each concentration. A time control was also measured for comparison. In all cases, the time controls showed no significant change in frequency. However, time-control amplitudes decreased significantly in most experiments over time. All statistical comparisons of amplitude were done by using the time controls.
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To determine whether there were major differences in L-Ca2+ activity between the jejunum and ileum, we conducted all experiments separately on muscle from each location. Application of 0.3, 1, and 3 µM nicardipine to ileum had the same effect as in the jejunum (n = 5) (Fig. 1), except for one important difference. The application of nicardipine to jejunal and ileal smooth muscle resulted in similar contraction frequencies, regardless of initial frequency. Faster-paced jejunal muscle, and slower paced ileal muscle all slowed to between 30 and 40 contractions/min after 3 µM of nicardipine was added. This occurred in LM and CM. A linear regression analysis showed the relationship between initial frequency and change in frequency (Fig. 3) for individual segments. In LM, we found an R2 of 0.88 (P 0.01) and a slope of 0.91, and in CM, a R2 of 0.86 (P
0.01) and a slope of 0.94 between the initial frequency and the change in frequency 15 min after 3 µM nicardipine. In comparison, there was no linear relationship found between initial and final frequencies (not shown).
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To determine whether L-Ca2+ channel activity in smooth muscle was directly responsible for the pacing gradient found throughout the intestines, we applied the L-Ca2+ channel agonist BAY K 8644 to muscle strips to determine whether pacing frequencies, particularly in the ileum, could be increased in this way. BAY K 8644 is a dihydropyridine, like nicardipine, but it acts as a L-Ca2+ channel agonist by increasing the mean open time of the channel (11, 33). Furthermore, BAY K 8644 and dihydropyridine antagonists like nicardipine are thought to interact allosterically, which could account for some of our results outlined below. We applied BAY K 8644 at an initial concentration of 107 M. After 10 min, we measured the frequency and amplitude and added more BAY K 8644 to a final concentration of 106 M. After 10 min, we again measured frequency and amplitude and added 1 µM nicardipine. This was to determine whether the effects of nicardipine could be blocked by prior addition of an L-Ca2+ channel agonist. Measurements were recorded 10 min after nicardpine application. These experiments were carried out in LM and CM strips from the jejunum and ileum (Fig. 4).
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Application of 107 M BAY K 8644 to ileal LM strips did not significantly affect frequency (n = 9). Subsequent application of 106 M BAY K 8644 increased frequency, but still well below the frequency of jejunum (n = 9) (Fig. 4). Subsequent application of 1 µM nicardipine did not significantly change the frequency (n = 4). BAY K 8644 also blocked the effects of nicardipine successfully in the ileum. However, different from jejunal muscle, application of 107 and 106 M BAY K 8644 to ileal LM caused an increase in contraction amplitude (n = 8) (Fig. 4). Subsequent application of nicardipine decreased the amplitudes back to control levels (n = 4). Application of BAY K 8644 (107 and 106 M) and nicardipine to ileal CM did not significantly change frequency, and amplitude was increased only after 106 M. Subsequent application of 1 µM nicardipine caused a slight decrease in contraction amplitude (n = 4) (Fig. 4). However, this decrease is much less than that observed with nicardipine alone. The major difference observed between jejunum and ileum was the occurrence of increased contraction amplitude in ileum after BAY K 8644. In both tissues (LM and CM), the actions of nicardipine were blocked by prior application of BAY K 8644, but BAY K 8644 had little or no effect on pacing frequency.
In a separate series of experiments, we added nicardipine before BAY K 8644 to determine whether BAY K 8644 could successfully reverse the effects. In all cases, BAY K 8644 was unable to reverse the effects of nicardipine (not shown).
W/W++ and W/WV Frequency Comparison
Strips from W/W++ mice were placed into muscle baths and their contraction frequencies were recorded after nerve block. Ileum LM and CM, having respective frequencies of 43.0 ± 1.0 and 39.3 ± 1.1 contractions/min, were found to be significantly slower than jejunal LM and CM, having frequencies of 49.6 ± 1.9 and 49.1 ± 0.9 (Fig. 5A). Furthermore, ileal LM had a higher average frequency than ileal CM. LM strips from W/WV mice were also studied, and jejunal LM was found to have a frequency of 50.2 ± 2.3 contractions/min (Fig. 5B), compared with ileal LM paced at an average frequency of 37.0 ± 1.5 contractions/min (P 0.0001, n = 21), similar to wild-type intestine despite the absence of ICC-MP.
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In this series of experiments, similar to previous experiments on Balb/C mice, nicardipine and BAY K 8644 were added to LM and CM muscle tissue preparations from W/W++ mice. These experiments successfully replicated those done previously in Balb/C tissue and described in Application of Nicardipine (not shown).
W/WV, Application of Nicardipine, and BAY K 8644
We have previously reported that W/WV LM displays robust pacing that is not susceptible to blockade by cyclopiazonic acid or nicardipine. In this study, we examined more closely the role of L-Ca2+ channels in W/WV LM pacing. Unlike wild-type tissue, application of 1 µM nicardipine to jejunal LM increased contraction frequency slightly (n = 8) (Fig. 6). Subsequent application of 106 M BAY K 8644 lowered frequency back down to control levels. Effects on amplitude were not different from previous experiments on Balb/C or W/W++ mice (Fig. 6).
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DISCUSSION |
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We found that the effects of nicardipine were dependent on concentration. Nicardipine (0.3 µM) had a smaller effect than 1 or 3 µM nicardipine, which were similar. Blockade of L-Ca2+ channels consistently reduced the frequency of pacing in intestinal smooth muscle to 3040 contractions/min. This is likely due to the effect on ICC-MP, because contractions remained regular although smaller, but effects on slow-wave transmission and smooth muscle contractibility may also contribute. The cycling of calcium among the cytosol, ER, and mitochondria has been suggested to be required for ICC pacing (34, 36). It is likely that L-Ca2+ channels are also important in this process, as a source of calcium, either directly or to refill Ca2+ stores, and/or a signal to trigger the release of calcium from intracellular stores. An L-Ca2+ channel, dihydropyridine-sensitive current, has been found to account for 50% of the voltage-dependent inward current in cultured ICC from the mouse small intestines (16). By influencing the influx of calcium through L-Ca2+ channels, the pacing mechanism can be regulated. For example, Jackson et al. (15) have found that an autoantibody, produced in type 1 diabetes, disrupts intestinal motility by activating smooth muscle L-Ca2+ channels at the dihydropyridine binding site. Furthermore, pacing abnormalities found in Crohn's disease and some inflammatory bowel disorders may be partly caused by decreased ICC or smooth muscle L-Ca2+ channel activity (17, 23).
The blockade of L-Ca2+ channels appeared to have large, direct effects on smooth muscles, observed as decreases in contraction amplitude. We cannot be sure that the effects of nicardipine on smooth muscle account for this, because an effect on slow-wave transmission to muscle might be involved, but it is the most likely explanation. As described in the introduction, slow waves in mouse intestine are not considered to be affected by block of L-Ca2+ channels, but action potentials are abolished. We have observed a high level of L-Ca2+ channel expression in both LM and CM in the mouse intestine as well as ICC, and nicardipine at 1 µM blocked KCl-induced contractions in LM and CM, which are independent of ICC function (unpublished observations). These results support previous experiments demonstrating the importance of L-Ca2+ channels in smooth muscle contractibility (2, 8, 10).
Frequency differentials in ICC pacing along the intestine are crucial for normal transit of gut contents. Typically the jejunum contracts at a higher frequency than the ileum (4, 32). Investigations in animals and humans have shown that electrical pacing at higher frequencies in distal sites drives the intestine slow waves in a reverse direction and delays or reverses transit. Thus the basis for the intestinal frequency gradients of slow waves is of major physiological significance (3, 9, 20, 29).
Our initial experiments suggested that the difference in pacing between the jejunum and ileum might be due to ICC-MP-based L-Ca2+ channel activity. From these results and previous reports that BAY K 8644 increases both cytosolic calcium levels in antral smooth muscle (26) and voltage-dependent inward currents in cultured ICC (16), we expected that the application of a L-Ca2+ channel agonist would increase pacing in the ileum to that of the jejunum. This was not the case. Instead, only a time-dependent decrease in pacing was prevented. However, the effects of nicardipine were reduced or completely blocked, indicating that BAY K 8644 was successfully agonizing the L-Ca2+ channels. Due to the inability of an L-Ca2+ channel agonist to increase pacing, our hypothesis that L-Ca2+ channels were alone responsible for the difference in pacing observed in the jejunum and ileum was weakened. Clearly, it is not simply differences in the level of activation of L-Ca2+ channels in different intestinal segments, which accounts for frequency differences. However, it remains certain that L-Ca2+ channel activity is required for intestinal muscle to be paced above a certain level.
In W/WV mouse intestine that does not have ICC-MP, we found LM muscle from the jejunum to be paced faster than the ileum, similar to wild-type tissue that has ICC-MP. This difference was originally thought to originate exclusively from ICC-MP, but this finding suggests that there is a conservation of the gradient of pacing frequency between W/WV and wild-type intestine that persists despite the absence of a network of ICC-MP. There are, however, major differences that exist between LM pacing in W/WV and wild-type intestine: they now include the lack of responses in frequencies of W/WV LM to SERCA pump and L-Ca2+ channel blockade (4). In this report, we also found that W/WV LM did not respond to L-Ca2+ channel block except in some instances in jejunal LM where pacing was found to increase after exposure to nicardipine. From these findings it is clear that pacing in W/WV LM is driven by a different pacemaker than in wild-type muscle. Possible sources for this pacemaker include serosal ICC, intramuscular ICC, and the LM itself.
We have observed a network of serosal ICC present in both wild-type and W/WV mouse intestine. Techniques currently used to isolate ICC for culture do not distinguish between various types of ICC or their location of origin (19). It is possible that serosal ICC may take on the role of pacemaker in W/WV intestine. This could account for the differences found, although it would also suggest that serosal ICC have a pacemaker mechanism that is drastically different than that of wild-type ICC-MP. So far, there is no information about the coupling of serosal ICC to LM, either structural or functional. The other possibility is that LM retains some pacemaker function, because LM originates from the same mesenchymal precursor cells as ICC-MP (18, 27, 39, 40).
In conclusion, we have demonstrated an important role for L-Ca2+ channels in pacing of mouse intestine. Most of the work done in this area has focused on cultured and dissected mouse intestine. Using intact muscle segments, we have found differences between jejunum and ileum that could not be studied by using current techniques of cell culture and dissection. A closer examination of the role of L-Ca2+ channels may help to uncover the basis for differences in spontaneous pacing found throughout the intestine. It also appears clear that pacing in W/WV LM is driven by a separate pacemaker. The nature of this pacemaker is not known, but the presence of intact networks of serosal ICC in W/WV intestine suggests they could be involved. Presently, ICC-MP have been the focus of study and the involvement of other ICC needs to be addressed.
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FOOTNOTES |
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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.
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REFERENCES |
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