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
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ABSTRACT |
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
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INTRODUCTION |
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.
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METHODS |
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.
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RESULTS |
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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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.
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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.
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Figure 4A
summarizes changes in the resting membrane potential
(
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 ( 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.
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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.
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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).
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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.
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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.
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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).
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DISCUSSION |
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.
 |
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