Voltage sensitivity of slow wave frequency in isolated circular muscle strips from guinea pig gastric antrum

S.-M. Huang1, S. Nakayama1, S. Iino2, and T. Tomita3

Departments of 1 Physiology and 2 Anatomy, School of Medicine, Nagoya University, Nagoya 466-8550; and 3 Division of Smooth Muscle Research, Institute of Medical Sciences, Fujita Health University, Toyoake, Aichi 470-11, Japan


    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

In circular muscle preparations isolated from the guinea pig gastric antrum, regular spontaneous electrical activity (slow waves) was recorded. Under normal conditions (6 mM K+), the frequency and shape of the slow waves were similar to those observed in ordinary stomach smooth muscle preparations. When the resting membrane potential was hyperpolarized and depolarized by changing the extracellular K+ concentration (2-18 mM), the frequency of slow waves decreased and increased, respectively. Application of cromakalim hyperpolarized the cell membrane and reduced the frequency of slow waves in a dose-dependent manner. Cromakalim (3 µM) hyperpolarized the membrane, and slow waves ceased in most preparations. In the presence of cromakalim, subsequent increases in the extracellular K+ concentration restored the frequency of slow waves accompanied by depolarization. Also, glibenclamide completely antagonized this effect of cromakalim. In smooth muscle strips containing both circular and longitudinal muscle layers, such changes in the slow wave frequency were not observed. It was concluded that the maneuver of isolating circular smooth muscle altered the voltage dependence of the slow wave frequency.

pacemaker; smooth muscle; voltage dependence; potassium channel opener


    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

SMOOTH MUSCLES IN the gastrointestinal tract show slow electrical oscillation, so-called "slow waves." The properties of slow waves are, however, different when they are rigorously investigated in numerous parts and species of the intestinal tract (28, 34).

Most slow waves seen in isolated smooth muscle tissues are hardly affected by blocking neuronal activity (e.g., with atropine and tetrodotoxin). They are thus considered to be myogenic (34). In canine colon, the amplitude of the slow waves is maximal near the submucosal surface, and they are not seen when the submucosal surface is removed (21, 29). Similarly, the myenteric plexus and deep muscular plexus have been reported to be important in generating slow waves in the small intestine (12, 30). These regions of the intestinal tract contain abundant interstitial cells of Cajal (4, 39) or specialized smooth muscle cells (19, 36). Thus it has been hypothesized that slow waves originate from these cells. On the other hand, the smooth muscle of guinea pig stomach has rather unique electrical properties. The frequency of slow waves is only slightly affected by changing the membrane potential with a sucrose gap apparatus (25), partition chamber (22), and K+ channel opening drugs (15, 17). Furthermore, even when the membrane potential was clamped, a regular current oscillation was observed (26).

In the present study, we used a circular muscle preparation isolated from the guinea pig gastric antrum, in which the longitudinal muscle layer and mucosa were removed. In this preparation, the frequency of slow waves was recorded with conventional microelectrodes and was clearly affected by changes in the resting membrane potential; depolarization increases, while hyperpolarization decreases the frequency. Furthermore, we found that slow waves in isolated circular muscle strips were suppressed by low concentrations (1-10 µM) of Ni2+. The isolation maneuver seems useful to investigate the underlying mechanisms of slow wave generation. Also, the altered characteristics of slow waves in isolated circular muscle strips of the guinea pig gastric antrum may correspond to the high membrane potential dependence of slow wave frequency seen in other species.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Guinea pigs (250-350 g) of either sex were killed by stunning and exsanguination. The stomach was excised and cut into two parts along the greater and lesser curvature. The mucosa was carefully removed. Small strips (~0.4 × 4-5 mm) were dissected along circular muscle layer from the thickest part (distal end) of the antrum pylori (see Fig. 1 in Ref. 31). The muscle layer in this region is thick enough to isolate circular smooth muscle alone. Circular muscle preparations were dissected with a pair of small scissors from the mucosal side of the antrum.

Electrical and mechanical activities were simultaneously measured from the muscle strips, using essentially the same methods as described previously (7, 14, 37), with a high input impedance amplifier (MEZ-8201, Nihon Koden) and a strain gauge (UL-10GL, Shinkoh). The recording chamber was warmed to 34°C, and the preparations were superfused (at a constant rate of 2 ml/min) with physiological saline solutions prewarmed at the same temperature. In the present experiments, the effects of changing the membrane potential on the frequency of spontaneous electrical activity were examined. To alter the membrane potential, the extracellular K+ concentration was changed or a K+ channel opener, cromakalim, was applied. The extracellular K+ was not completely removed, because such treatments would cause depolarization of the cell membrane (35) and would also alter the intracellular ionic environments in smooth muscle (23). The length of the preparation was too short to apply the sucrose gap (25) or the partition chamber technique (1).

After electrical and mechanical recordings had been done, some of the muscle strips were fixed with 2.5% glutaraldehyde followed by 1.0% osmium tetroxide. The specimens were then dehydrated with graded ethanol and embedded in epoxy resin. One-micrometer-thick sections were stained with 1.0% toluidine blue and examined under a light microscope (19).

The normal solution had the following composition (in mM): 127 NaCl, 6 KHCO3, 2.4 CaCl2, 1.2 MgCl2, 11.8 glucose, and 10 Tris-Cl, with pH adjusted to 7.4 at 34°C. The K+ composition was modified by isosmotically adjusting Na+. Cromakalim was a generous gift from SmithKline Beecham Laboratories. Glibenclamide was purchased from Sigma (St. Louis, MO). Tetraethylammonium (TEA)-Cl and CdCl2 were from Wako Pure Chemical Industries (Tokyo, Japan). NiCl2 was from Katayama Chemical Industries (Nagoya, Japan). The stock solutions (10 mM) of cromakalim and glibenclamide were made by dissolving them in ethanol and dimethyl sulfoxide, respectively. Nifedipine (Sigma) was dissolved in ethanol. Its stock solution (1 mM) was protected from light and kept cool.

Numerical data are expressed as means ± SD. Differences between means were evaluated by paired t-tests, and a P value of <0.05 was taken as statistically significant.


    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Slow waves in isolated circular muscle preparations. Effects of changing the membrane potential on slow wave frequency were examined in circular smooth muscle strips isolated from the guinea pig gastric antrum. In previous experiments, we normally isolated smooth muscle strips from the oral half of the antrum. On the other hand, the distal end of the antrum pylori has thicker muscle layer and allowed us relatively easily to isolate the circular muscle layer alone (by dissecting with small scissors from the mucosal side). Thus we used this part of the gastric antrum in the present study.

When the circular muscle preparations were superfused with normal solution, regular spontaneous electrical activity (slow waves) was found to be at 3-6 cycles/min over 100 preparations. The slow wave frequency was within the range previously reported (7, 14, 25). In whole muscle layer preparations, it has been shown that slow waves are divided into two components. The early phase is called the first component, and some of the whole muscle preparations show a typical first component that is distinguishable as a trapezoid-like potential of ~10 mV amplitude (25, 34). Despite variations in the shape of slow waves, the typical first components were not observed in the preparations used in this study (circular muscle preparations).

In five stomachs, characteristics of slow waves were more carefully compared using paired whole muscle layer (Fig. 1B) and isolated circular muscle preparations (Fig. 1C). After slow waves in a whole muscle preparation (both circular and longitudinal muscle layers being present) were recorded under superfusion with normal solution, a circular muscle strip was isolated from the whole muscle preparation and then mounted in the same recording chamber. After ~1 h, the slow wave frequency in such circular muscle preparations (4.3 ± 0.2 cycles/min) was slightly less, but not statistically (P < 0.05) significantly different from that of the control whole muscle preparations (4.9 ± 0.2 cycles/min). Also, the amplitude of slow wave and duration of half-maximal amplitude were not significantly changed by this isolation maneuver (Table 1).


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Fig. 1.   Pen recordings of electrical activities in stomach smooth muscle preparations. A: corpus circular muscle in which longitudinal muscle was isolated from serosal side was used. B: an intact whole muscle layer preparation obtained from the distal end of the antrum. From preparation shown in B, a circular muscle strip was dissected, and, after the muscle strip was equilibrated for ~1 h, traces in C were recorded. Mucosa had been removed in all 3 preparations (A-C). Cromakalim (10 µM) was applied during period indicated by bars. Resting membrane potential levels in A-C were approximately -61, -68, and -66 mV, respectively.

                              
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Table 1.   Comparison of slow waves between paired whole muscle and isolated circular muscle preparations

In Fig. 1A, a circular muscle preparation was obtained from the corpus region of the guinea pig stomach. The muscle layer was thin in this region; thus longitudinal muscle was carefully removed from the serosal side with a fine pair of forceps. In circular muscle strips prepared from this region, the slow wave frequency was not affected upon hyperpolarization induced by 10 µM cromakalim in the majority of the experiments, while the amplitude of slow wave was often reduced, as shown in Fig. 1A. Also, in some experiments using this type of preparation, slow waves ceased upon hyperpolarization. These responses were essentially the same as previously observed (17). On the other hand, in circular muscle strips isolated from the thickest part (distal end) of the gastric antrum, membrane hyperpolarizations consistently affected the slow wave frequency (Fig. 1C). Thus, from the next section, effects of changing the membrane potential on slow wave frequency were quantified in circular muscle preparations isolated from this region. Please note that before the longitudinal muscle layer (and myenteric plexus) was removed, experimentally induced hyperpolarization did not alter the frequency in the same muscle preparation (Fig. 1B).

Figure 2 shows photographs of smooth muscle preparations stained with toluidine blue. The cross sections shown in Fig. 2, A-C, are the same types of preparations used in Fig. 1, C, B, and A, respectively. The histological examination revealed that in the circular muscle preparations obtained from the antrum region (Fig. 2A), only the circular muscle layer and a part of submucosa remained. On the other hand, the circular muscle preparations obtained from the corpus region (Fig. 2C) contained a part of myenteric plexus as well. Also, this type of preparation sometimes contained a part of longitudinal muscle layer.


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Fig. 2.   Photographs of stomach smooth muscle preparations stained with toluidine blue. Cross sections shown in A and B were obtained from gastric antrum, whereas those in C and D are from corpus region. A and C are circular muscle preparations, whereas B and D are whole muscle preparations. Mucosa was removed with scissors in all preparations. Note that circular muscle preparation obtained from gastric antrum (A) was dissected from mucosal side with a fine pair of scissors; on the other hand, that obtained from corpus was prepared by removing longitudinal muscle layer with fine forceps. (Smooth muscle in gastric antrum was thick enough to dissect with scissors.) Cross sections shown in A-C were obtained from same types of smooth muscle preparations used in Fig. 1, C, B, and A, respectively. Myenteric ganglion, myenteric border, and submucosal border are indicated by asterisk, arrow, and arrowhead, respectively. CM and LM are circular and longitudinal muscle layers, respectively. Scale bar, 100 µm.

Effects of changing extracellular K+ concentration. First, the cell membranes were hyperpolarized or depolarized by changing the extracellular K+ concentration in isolated circular muscle preparations. Figure 3 shows an example of such an experiment. The normal solution contained 6 mM K+. After control electrical activity was observed, the extracellular K+ concentration was changed to various concentrations (2-18 mM). The resting membrane potential (the most hyperpolarized potential during slow wave oscillation) was hyperpolarized by decreasing the K+ concentration and was depolarized by increasing the K+ concentration in a dose-dependent manner. When the cell membrane was hyperpolarized, the frequency of the slow waves decreased and vice versa. Under membrane hyperpolarization, premature spontaneous depolarizations without overshoot sometimes occurred (data not shown).


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Fig. 3.   Representative pen recordings of electrical activity upon changing extracellular K+ concentration. After observation of control spontaneous electrical activity (slow waves) in normal solution (6 mM K+), extracellular K+ concentration was changed to 2, 4, 9, 12, 15, or 18 mM. Recordings shown were obtained in same preparation. At beginning of series of experiments, resting membrane potential was approximately -68 mV.

Figure 4A summarizes changes in the resting membrane potential (Delta V, in mV) and slow wave frequency (in cycles/min) when the extracellular K+ concentration was altered. After the effects of changing the K+ concentration had stabilized, the frequency was estimated by averaging three consequent slow waves. Figure 4B shows the correlation of these two parameters. The frequency is expressed relatively, taking the control value to be one. A clear voltage-dependent change in the frequency of slow waves was seen. When the resting membrane potential was changed from -3.7 to +16.0 mV, the frequency correspondingly changed approximately from 40 to 160% of the control, respectively.


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Fig. 4.   Effects of changing extracellular K+ concentration on membrane potential and frequency of slow waves. A: changes in resting membrane potential (Delta V, in mV; solid bars) and frequency of slow waves (in cycles/min; gray bars) are shown. Extracellular K+ concentration was changed from normal (6 mM) to various concentrations (2-18 mM). Gray bar shown at 6 mM K+ corresponds to frequency of slow waves seen in control (before changing extracellular K+ concentration). Error bars show ±SD; number of experiments is indicated in parentheses. B: correlation plot of changes in membrane potential and frequency of slow waves. Frequency of slow waves is normalized with control value. Mean value obtained in each K+ concentration is plotted with ±SD.

Effects of cromakalim. Cromakalim is known to hyperpolarize the cell membrane due to activation of ATP-sensitive K+ channels (inhibited by glibenclamide) in smooth muscles (reviewed in Ref. 8) including guinea pig stomach (17) and other intestinal smooth muscles (9, 27). Effects of hyperpolarization induced by cromakalim on slow wave generation were examined in isolated circular muscle preparations (Fig. 5). Cromakalim hyperpolarized the membrane potential in a concentration-dependent manner (Fig. 6A), as expected from its K+ channel opening effect. In this treatment too, hyperpolarization clearly decreased the frequency of slow waves. With 3 µM cromakalim, the cell membrane was hyperpolarized by 4.8 ± 1.1 mV (n = 25), and slow waves ceased in 17 of 25 preparations (Fig. 5C). Subsequent washouts of cromakalim gradually restored the membrane potential, and slow waves appeared again as the resting membrane potential recovered. In all 17 experiments in which slow waves were eliminated by application of 3 µM cromakalim, the same phenomenon was observed. (The analysis shown in Fig. 6 was done using the remaining eight preparations.) These results are in contrast to the fact that the frequency of slow waves is not affected by membrane hyperpolarization induced by cromakalim in stomach smooth muscle preparations containing both muscle layers (17).


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Fig. 5.   Effects of cromakalim on electrical activity. After observation of control electrical activity, various concentrations (0.3-3 µM) of cromakalim were applied for ~100 s. Resting membrane potential was approximately -66 mV.


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Fig. 6.   Effects of changing membrane potential by application of cromakalim. A: changes in resting membrane potential (solid bars) and frequency of slow wave (gray bars) are shown. Various concentrations (0.3-3 µM) of cromakalim (CRM) were used after observing control slow waves. Error bars show ±SD; number of experiments is indicated in parentheses. B: correlation plot of changes in membrane potential and frequency of slow waves normalized with control value. Mean value obtained in each concentration of cromakalim is plotted with ±SD. Depolarization and corresponding increase in frequency caused by increasing extracellular K+ concentration to 12 mM in presence of cromakalim (3 µM) are also plotted.

In Fig. 6A, effects of cromakalim (0.3-3 µM) on the membrane potential and frequency of slow wave are summarized. Both parameters were decreased by application of cromakalim in a dose-dependent manner. In Fig. 6B, the correlation between the membrane potential and slow wave frequency during application of cromakalim is plotted. With the use of this drug, a clear voltage-dependent change in the slow wave frequency was also seen.

Figure 7 shows antagonistic effects of glibenclamide on cromakalim-induced hyperpolarization. In Fig. 7A, the control response was obtained by application of 5 µM cromakalim; slow waves ceased and were accompanied by hyperpolarization. On the other hand, application of glibenclamide (1 µM) alone had little effect on either resting membrane potential or shape of slow waves (Fig. 7B). When cromakalim (5 µM) was applied, 2.5 min after application of glibenclamide (Fig. 7C), the effects of cromakalim were completely antagonized (spontaneous mechanical activity was also preserved in the presence of glibenclamide, data not shown). These results suggest that in circular muscle preparations, the cessation of slow wave, observed during application of cromakalim, is due to hyperpolarization induced by activation of K+ conductance, which is sensitive to glibenclamide (8).


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Fig. 7.   Antagonism of glibenclamide on cromakalim-induced changes in electrical activity. A: control response to 5 µM cromakalim. Application of 1 µM glibenclamide alone hardly affected spontaneous electrical activity (B). In presence of glibenclamide (1 µM), cromakalim (5 µM) induced neither hyperpolarization nor suppression of spontaneous activity (C).

Interaction of cromakalim and extracellular K+. Interaction of cromakalim and extracellular K+-induced changes in membrane potential on slow waves was examined (Fig. 8). Mechanical (Fig. 8, top traces) and electrical responses (Fig. 8, bottom traces) were recorded simultaneously. After a control response to 3 µM cromakalim (2-min application) was observed (Fig. 8A), the interaction of cromakalim and extracellular K+ was tested. In Fig. 8B, after cromakalim had hyperpolarized the cell membrane by ~6 mV (~1 min after application of cromakalim), the extracellular K+ concentration was increased to 12 mM. This treatment depolarized the cell membrane and restored slow wave activity. However, the mechanical response induced by the slow waves was still suppressed (although a small contraction was observed during application of 12 mM K+). The subsequent normalization of the extracellular K+ concentration (to 6 mM) reversed the membrane potential change and eliminated the slow waves again. In some of the preparations in which the same protocol was applied, phasic contractions accompanied by slow waves were observed during exposure to a high-K+ solution in the presence of cromakalim, although the recovery of the amplitude was incomplete.


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Fig. 8.   Recovery of spontaneous activity by increasing extracellular K+ concentration and by application of tetraethylammonium (TEA). Mechanical (top traces) and electrical activities (bottom traces) to an application of 3 µM cromakalim are shown. A: control response. B: recovery of spontaneous electrical activity by increasing extracellular K+ concentration in presence of cromakalim. C: 20 mM TEA was applied.

The recovery of the frequency of slow waves upon increasing the extracellular K+ concentration (to 12 mM) was also plotted in Fig. 6, A and B (n = 5). When the extracellular K+ was increased to 12 mM in the presence of cromakalim (3 µM), the cell membrane was relatively depolarized by 5.0 ± 1.5 mV compared with control resting membrane potential, and the frequency of slow waves was 20% greater than that of control. The degree of depolarization caused by 12 mM K+ in the presence of cromakalim (Fig. 6B) was smaller than that with 12 mM K+ in the absence of cromakalim (Fig. 4B) and was rather similar to that with 9 mM K+. With comparison of Figs. 4B and 6B, the changes in the frequency of slow waves seemed to be comparable when the membrane potential was manipulated to similar degrees, regardless of the extracellular K+ concentration or application of K+ channel opener.

Recovery of slow waves during application of cromakalim was also achieved by 20 mM TEA (Fig. 8C), but, in contrast to increasing the extracellular K+ concentration, this treatment restored mechanical responses as well. The contraction was usually larger than the control; also, the initial spike was much larger and slow waves often possessed late spikes on the plateau phase.

Effects of cation channel blockers on slow waves. In isolated circular muscle, underlying ionic conductance for slow wave generation was further investigated using cation channel blockers. In Fig. 9A, the upper and lower continuous pen recordings show mechanical and electrical spontaneous activities, respectively. Neither was affected by application of 0.3 µM tetrodotoxin (TTX). The expanded electrical activies were recorded before (Fig. 9A, trace a) and after applications of 0.3 (Fig. 9A, trace b) and 1 µM TTX (Fig. 9A, trace c). These results suggest that voltage-sensitive Na+ channels do not play a dominant role in this smooth muscle tissue, like the majority of other smooth muscles (34).


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Fig. 9.   Effects of tetrodotoxin (TTX; A) and nifedipine (B) on spontaneous activity. A: example of effects of TTX. Top and bottom continuous pen recording traces show mechanical and electrical activities, respectively. TTX (0.3 µM) was applied for ~2 min. Expanded spontaneous electrical activity is shown below continuous recordings. Trace a shows before application of TTX (control). Traces b and c were obtained during applications of 0.3 and 1 µM TTX, respectively. Effects of nifedipine are shown in B. Like in A, top and bottom continuous recordings show mechanical and electrical recordings, respectively. Spontaneous electrical activity is shown expanded. Trace a, control; traces b-e, in presence of 0.3, 1, 3, and 10 µM nifedipine, respectively. Resting membrane potentials in A and B were approximately -68 and -70 mV, respectively.

Nifedipine is known to block L-type Ca2+ channels, the presence of which have already been shown in guinea pig stomach smooth muscle (18). Figure 9B shows an example of the effects of nifedipine (0.3 µM) on mechanical and electrical activities. The tension development was completely inhibited (Fig. 9B, top trace). In contrast, neither the resting membrane potential nor the frequency of slow waves was affected by nifedipine (Fig. 9B, bottom trace). Electrical activities obtained from the same preparations are shown expanded. The shape of the slow waves showed little change with applications of nifedipine, up to 3 µM (Fig. 9B, traces b-d). Nifedipine (10 µM) slightly reduced the amplitude of slow waves (Fig. 9B, trace d). Similar results were obtained in five other preparations. In one preparation, 10 µM nifedipine significantly depressed the late plateau phase of slow wave by ~50%. In some of the circular muscle preparations, secondary spikes were observed on the plateau phase of the slow wave under normal conditions. Nifedipine (1-3 µM) selectively depressed these spike activities. This result agrees well with the effects of nifedipine reported in canine gastric smooth muscle (13).

The continuous pen recording in Fig. 10 shows the effect of (1 µM) Ni2+, which is often used to block T-type voltage-sensitive Ca2+ channels (11, 40). Short-term applications of Ni2+ suppressed slow waves (mainly in the late plateau phase), and, in 9 of 14 preparations, this treatment slightly (by up to 2-3 mV) and transiently hyperpolarized the cell membrane. However, suppression of the plateau phase did not correlate with the degree of hyperpolarization, but recovery of the plateau phase was much slower than that of the resting membrane potential. Also, the slow wave frequency tended to slightly increase with Ni2+ (to 122 ± 16% with 3 µM, n = 6), irrespective of the small changes in the resting potential. The expanded traces (Fig. 10, traces a-c) show effects of 1, 3, and 10 µM Ni2+, respectively. As shown in Fig. 10, trace c, 10 µM Ni2+ almost fully abolished slow waves (n = 5). (In all preparations, the amplitude of slow wave decreased to below 10% of control.) Similar complete suppression of slow wave was also caused by 10 µM Cd2+. Figure 11 shows effects of high K+ in the continuous presence of 10 µM Ni2+. When the extracellular K+ concentration was increased to 12 mM, the cell membrane (resting potential) was depolarized by ~6 mV and restored slow wave activity. However, the slow wave frequency was less compared with control.


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Fig. 10.   Effects of Ni2+ on spontaneous electrical activity. Top trace, spontaneous electrical activity was continuously recorded, and 1 µM Ni2+ was applied for ~2 min. Expanded traces a-c show effects of 1, 3, and 10 µM Ni2+, respectively. Resting membrane potential was approximately -66 mV.


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Fig. 11.   Incomplete recovery of slow waves by increasing extracellular K+ concentration in presence of Ni2+. Top and bottom traces show mechanical and electrical activities, respectively. During continuous exposure to 10 µM Ni2+, extracellular K+ concentration was increased from 6 to 12 mM (for ~4 min).


    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

In smooth muscle strips isolated from the guinea pig gastric antrum, the frequency of spontaneous electrical activity (slow waves) is only slightly (15-20%) affected by manipulating the resting membrane potential when accomplished using a sucrose gap apparatus (25). This is also true when the membrane potential is changed using a partitioned chamber (22), and when the cell membrane is hyperpolarized with cromakalim (15, 17). The cromakalim-induced hyperpolarization is presumably due to activation of the so-called ATP-sensitive K+ channels (2, 3).

In the present study, we used circular smooth muscle preparations from which longitudinal muscle and myenteric plexus were removed (although a part of submucosa remained) and examined effects of changing the membrane potential on slow waves. To make such circular muscle preparations relatively easily, we used the thickest part (distal end) of the gastric antrum. On the other hand, in previous experiments the membrane potential was usually recorded in the proximal half of the antrum and corpus regions where the smooth muscle layer is much thinner. The circular muscle preparations used in the present study also show regular spontaneous electrical activity (slow waves) accompanied by contraction, and the frequency (3-6 cycles/min) and shape of slow waves were within the range previously observed (7, 14). Furthermore, as shown in Fig. 1B, before isolation procedure, whole muscle layer preparations obtained from the distal end of the gastric antrum also possess the characteristic feature seen in other parts of guinea pig stomach smooth muscle; the slow wave frequency was little changed during application of cromakalim.

In the circular muscle preparations, nifedipine, a dihydropyridine Ca2+ channel antagonists, up to 10 µM affected neither the shape (except for blocking the late spikes) nor frequency of slow waves, although the corresponding mechanical activity was completely inhibited by 0.3 µM of this drug (Fig. 9). This result is also consistent with the previous observations made in whole muscle layer preparations using Ca2+ channel antagonists [verapamil, Golenhofen and Lammel (10); nifedipine, personal communication from M. Tsugeno], suggesting that dihydropyridine-sensitive (L-type) Ca2+ channels play the same role even after isolating circular muscle layer alone, i.e., contraction is produced by depolarization-mediated Ca2+ influx. Also, the present observation that TTX affects neither mechanical nor electrical spontaneous activities (Fig. 9A) is consistent with the previous result obtained in the whole muscle layer preparations (25).

Although there are many similar features of spontaneous activity observed in the isolated circular muscle used in the present study and previous preparations, we found in the isolated circular muscle preparations that the frequency of slow waves is significantly altered by changing the membrane potential with either application of various concentrations of K+ or cromakalim. Furthermore, applications of cromakalim greater than 3 µM abolished slow wave generation accompanied by membrane hyperpolarizations. This is in contrast to the previous observation that in whole muscle layer preparations the frequency of slow waves was negligibly affected by cromakalim up to 3 µM and that even when 10 µM cromakalim was applied, slow waves never ceased in spite of significant hyperpolarizations (17).

During application of cromakalim, when the negativity of the resting membrane potential was returned to control levels by increasing the extracellular K+ concentration, the frequency of electrical slow wave fully restored, but its shape was different from control slow wave (Fig. 8B); it had a small action potential with large spike activity. Also, the recovery of contraction with high K+ is incomplete. On the other hand, when TEA was applied in the presence of cromakalim, large contractions accompanied by potentiated action potential and spike activity are observed (Fig. 8C). When cromakalim was applied in the presence of glibenclamide, both electrical (Fig. 7) and mechanical activity (data not shown) were little affected. These results suggest that the shape (size) of slow wave as well as spike activity is important in excitation-contraction coupling in smooth muscle of the guinea pig gastric antrum.

Ni2+ is often used to block T-type Ca2+ channels, which play an important role in generating spontaneous electrical oscillation in cardiac pacemaker cells (11). In canine colonic smooth muscle, it has been shown that slow wave activity is significantly affected by applications of Ni2+ (40). At 1 mM, Ni2+ abolished slow waves by initially slowing the velocity of the upstroke component (initial spike) with relatively low concentrations of Ni2+ (50% reduction by 40 µM). In the present study, we also examined effects of this inorganic Ca2+ channel blocker in the isolated circular muscle preparations of guinea pig stomach and found that even 10 µM Ni2+ almost fully suppressed slow waves. The inhibitory effect of Ni2+ in this preparation is more prominent on the (late) plateau phase (Fig. 10B). This high sensitivity to Ni2+ could be used to identify pacemaker cells in isolated circular muscle; however, the underlying mechanisms (ionic conductances) to generate slow waves may differ among tissues. In guinea pig stomach, Ni2+ might suppress slow waves via metabolic inhibition; iodoacetic acid, a metabolic inhibitor, also causes suppression of the late plateau phase before elimination of slow wave activity (22). Furthermore, like for Ni2+, low concentrations of Cd2+ suppressed slow waves in isolated circular muscle preparations in the same manner. Nonspecific block on some cation conductances may be responsible for the suppression of the plateau phase.

In the intestinal tract, it has been proposed that slow waves originate from the interstitial cells of Cajal (ICC) (32) that are localized in the myenteric plexus region and submucosal surface. The interstitial cells may be reevaluated as specialized smooth muscle cells (19, 24, 36). Patch-clamp techniques have revealed that isolated ICC produce regenerative electrical events in a voltage-dependent manner and spontaneously generate slow wave-like events under current-clamp conditions (20). On the other hand, molecular biological studies have recently revealed that in murine intestine the ICC express c-Kit proteinlike immunoreactivity and that slow waves are not seen in the murine intestinal tracts when development of the c-Kit-positive cells is prevented by mutation of the protooncogene c-kit (38). More recently, under voltage-clamped conditions, spontaneous electric current oscillations have been recorded in c-Kit-positive cells isolated from mouse intestine (33). The discrepancy between the electrical properties seen in these isolated cells can prompt us to postulate that there are multiple types of pacemaker cells used to generate slow waves. This hypothesis may explain the observations in the present study, i.e., voltage-sensitive and less sensitive pacemakers may exist in guinea pig gastric antrum, and in circular muscle preparations the latter pacemaker cells may be dominant.

In the guinea pig gastric antrum, it was suggested that the slow wave is divided into two parts: the first (voltage-insensitive) and second (voltage-sensitive) components. A typical first component is the early part of slow wave, trapezoid-like shaped, and a following plateaulike potential is called the second component. However, in many recordings, the first component of the slow wave is often masked by the second component, and thus it is difficult to distinguish the first component in shape. On the other hand, in this tissue, two types of c-Kit-positive cells have been shown: 1) spindle-shaped intramusclar interstitial cells (IC-IM) and 2) myenteric region interstitial cells (IC-MY) that possess multiple thin extensions and form a dense network (5). Furthermore, in the murine gastric antrum, it has been suggested that these two types of interstitial cells contribute to different functions (6). Some of the isolated circular muscle preparations used in the present study were histologically examined, and it was found that the myenteric plexus did not remain in those preparations. Our isolated circular muscle preparations contained only one type of interstitial cell (IC-IM), if any. Taken together, it seems likely that IC-MY in the guinea pig gastric antrum is a key to solving the alteration of voltage sensitivity after isolation maneuver. However, the present study does not directly address the roles of interstitial cells. There are many other possible accounts for the present results, e.g., IC-MY cooperates with other components of myenteric plexus to generate voltage-insensitive slow waves. Although such accounts are taken into consideration, the present study suggests that isolated circular muscle in the guinea pig gastric antrum, where only one type of interstitial cell remains, can produce voltage-sensitive slow waves the shape of which is not distinguishable from that of ordinary slow waves.

In conclusion, the voltage sensitivity of the slow wave frequency in guinea pig gastric antrum is modulated by isolating the circular smooth muscle layer. The altered characteristics of slow waves in this isolated circular muscle preparation may correspond to the high membrane potential dependence of the slow wave frequency seen in other species.


    ACKNOWLEDGEMENTS

We are grateful to Professors S. Kobayashi and K. Kuba and Drs. S. Torihashi and M. Tsugeno (Nagoya University) as well as Drs. L. M. Smith and J. F. Clark and Professor A. F. Brading (Oxford University) for invaluable information, stimulating discussion, and proofreading of this manuscript.


    FOOTNOTES

This work was partly supported by a grant-in-aid from the Ministry of Education, Science, and Culture, Japan.

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. §1734 solely to indicate this fact.

Address for reprint requests: S. Nakayama, Dept. of Physiology, School of Medicine, Nagoya University, Tsuruma 65, Showa-ku, Nagoya 466-8550, Japan.

Received 13 May 1998; accepted in final form 22 October 1998.


    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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