Intracellular recordings were made from isolated circular muscle bundles of rat gastric fundus. The majority of cells generated an ongoing discharge of electrical activity that were ≤10 mV in amplitude (unitary potentials). A second pattern of electrical activity was recorded in less than 1% of all impalements. This electrical activity was characterized by high frequency, large amplitude spontaneous transient depolarizations (STDs) with a maximum rate of rise (dV/dtmax) of 0.5 V/s. Injection of the fluorescent dye propidium iodide into cells and double labeling with an antibody against the Kit receptor revealed that unitary potentials were recorded from circular smooth muscle cells (CSMC), whereas STDs were generated by intramuscular interstitial cells of Cajal (ICC-IM). Sustained injection periods (>15 min) resulted in the spread of dye between CSMC, between ICC-IM, and between CSMC and ICC-IM. Two types of STDs were observed, regularly occurring continuous STDs and irregular noisy bursting STDs. The amplitude of STDs varied between the two types of STDs. Single units summed to develop STDs with a maximum amplitude of 30 mV. Sodium nitroprusside (3 μM) induced membrane hyperpolarization and abolished unitary potentials generated by CSMC. In contrast, the amplitude of STDs generated by ICC-IM was increased with membrane hyperpolarization. Hyperpolarization induced by pinacidil (10 μM) also increased the amplitude of STDs and enhanced dV/dtmax. These observations indicate that STDs generated in ICC-IM spread passively to the adjacent CSMC to evoke the discharge of unitary potentials in the gastric fundus.
- intramuscular interstitial cells of Cajal
- smooth muscle
- unitary potentials
interstitial cells of cajal (ICC) are distributed throughout the gastrointestinal (GI) tract from esophagus to internal anal sphincter (23, 25, 30). To date, morphological studies have identified several subtypes of ICC, including ICC lying between the smooth muscle layers at the level of myenteric plexus (ICC-MY or ICC-MP), ICC located within the smooth muscle layers and scattered amongst the smooth muscle cells (ICC-IM or ICC-CM, ICC-LM, for circular and longitudinal muscle layers, respectively), ICC associated with the deep muscular plexus located between the inner and outer circular muscle sublayers in the small intestine (ICC-DMP), and ICC found at the interface between the submucosa and circular muscle layer in the colon (ICC-SM or ICC-SMP) (3, 8, 23). In terms of their physiological function, ICC are generally classified into two main groups. 1) ICC-MY and submucosal ICC (ICC-SM) serve as pacemaker cells, and 2) intramuscular ICC (ICC-IM) and deep muscular plexus ICC (ICC-DMP) serve as mediators of neuronal input (25, 26). ICC-MY of the mouse small intestine have been most extensively studied. Reduced ICC-MY in the small intestine of W/Wv mice is associated with loss of slow waves (14, 34). In situ, ICC-MY of the small intestine generate rapidly, rising large potential changes (pacemaker potentials) (19, 20), and these events conduct, with decrement to intestinal smooth muscle cells (20).
Two subtypes of ICC are involved in the generation of slow waves within the circular muscle of the gastric antrum (31). These slow waves consist of two components; the first component is formed by electrotonic propagation of pacemaker potentials produced in ICC-MY, and the second component is formed by regenerative potentials produced in ICC-IM (6, 13). Recent work has demonstrated that corporal ICC-IM trigger the dominant pacemaker activity in the whole stomach by generating a regular high-frequency discharge of corporal slow waves, that antral ICC-MY conduct waves of depolarization initiated by corporal ICC-IM through the ICC-MY network, and that antral ICC-IM amplify the depolarization (the first component of antral slow waves) conducted by ICC-MY, giving rise to the second component of antral slow waves (13). Therefore, despite the very similar morphological properties (27), the functional roles of corporal and antral ICC-IM differ.
In contrast to the corpus and antrum, slow waves are not generated in or propagated to the fundus, and this region is typically referred to as electrically quiescent (21, 36). Lack of slow waves is thought to be attributable to the absence of ICC-MY in this region of the stomach (2, 23, 27). Most studies of ICC-IM in the fundus have centered on their role in enteric motor neurotransmission (2, 21, 35, 36). In fact, the fundus is not electrically quiescent and is characterized by an ongoing discharge of unitary potentials, which are small-amplitude, noise-like events in strips of muscle and larger amplitude events in tiny bundles of muscle (1, 2). Loss of ICC-IM in W/WV mice results in loss of unitary potentials (1, 2, 35), suggesting that the ICC-IM are the source of this spontaneous activity. The inability of unitary potentials to entrain and generate slow waves in the fundus appears to be attributable to the loss of a voltage-dependent mechanism that entrains unitary potentials in the antrum (1). The stochastic discharge of unitary potentials represents an excitatory mechanism in fundus muscles, imposing a more depolarized basal state on smooth muscle cells that falls within the window-current range for L-type Ca2+ channels. Thus unitary potentials are likely to contribute to the generation of basal tone in the fundus.
The electrical properties of ICC-IM have not been directly studied. Most of what is known about ICC-IM has been deduced from studies of unitary potentials in small bundles of circular muscle cells that contain a few ICC-IM (1, 7, 12, 28). The present study was designed to investigate the spontaneous electrical activity of circular muscles of the rat gastric fundus with intracellular microelectrodes. Fluorescent dye injection during impalements revealed that low-amplitude unitary potentials were recorded from circular smooth muscle cells (CSMC) and that large-amplitude spontaneous transient depolarizations (STDs) were recorded from bipolar cells with two primary processes. These morphological features of cells that generate STDs were identical to ICC-IM. On the basis of the similarities and dissimilarities between STDs and unitary potentials, the mechanisms underlying the generation of STDs and unitary potentials in gastric fundus tissues are discussed.
MATERIALS AND METHODS
Wistar rats of either sex, aged 8–12 wk, were anesthetized with fluoromethyl 2,2,2-trifluoro-1-(trifluoromethyl) ethyl ether (sevoflurane; Maruishi Pharmaceutical, Osaka, Japan) and euthanized by cervical dislocation and exsanguination. The use and treatment of animals was approved by the Institutional Animal Use and Care Committee at the Nagoya City University. Segments of gastric fundus were removed from animals and opened along the lesser curvature, in Krebs solution (see below). The mucosal layers, the serosal layers, and the longitudinal layers were carefully removed under a dissecting microscope. A tissue segment (0.2–0.4 mm wide and 1.0–1.5 mm long) was pinned out on a silicone rubber plate with the longitudinal side uppermost, and the plate was fixed at the bottom of an organ bath (8 mm wide, 8 mm deep, 20 mm long). The tissue was superfused with warmed (35°C) and oxygenated Krebs solution, at a constant flow rate of ∼2 ml/min. Because L-type Ca channels are not involved in the generation of unitary potentials recorded from mouse fundus (1), experiments were carried out in the presence of 3 μM nifedipine throughout to minimize the movement of muscles.
Conventional microelectrode techniques were used to record intracellular electrical activity from smooth muscle tissues, and the glass capillary microelectrodes (OD 1.5 mm, ID 0.86 mm; Hilgenberg, Malsfeld, Germany) filled with 2 M KCl had tip resistances ranging between 50 and 80 MΩ. Electrical responses recorded via a high-input impedance amplifier (Axoclamp-2B; Axon Instruments, Foster City, CA) were displayed on a cathode-ray oscilloscope (SS-7602; Iwatsu, Osaka, Japan) and stored on a computer for subsequent analysis and display.
To identify the morphological features of cells in the fundus, impalements were made with microelectrodes filled with 2 M KCl and 0.5% wt/vol propidium iodide (Sigma, St. Louis, MO). Impaled cells were labeled with propidium iodide by passing hyperpolarizing current pulses (duration 100 ms, intensity 1 nA, frequency 3 Hz for 5–30 min) supplied from an electric stimulator (SEN-3301; Nihon Kohden, Tokyo, Japan). After the cells were filled, the muscles were fixed overnight at 4°C with fresh 4% wt/vol paraformaldehyde in 0.1 M PBS. After fixation, the muscles were washed several times with PBS, mounted in DAKO fluorescent mounting medium (DAKO, Carpinteria, CA), covered with a coverslip, and viewed with a confocal microscope (LSM5 PASCAL; Carl Zeiss, Jena, Germany). The confocal microscope with a krypton-argon laser allowed the visualization of propidium iodide (488-nm excitation filter and 560-nm emission long-pass filter).
Kit immunohistochemical studies were carried out on whole mount preparations from the gastric fundus. Tissues were stretched to 110% of their resting length and width before being fixed in paraformaldehyde (4% wt/vol in 0.1 M phosphate buffer) for 30 min at 4°C. After fixation, whole mounts were washed in PBS (0.01 M, overnight) before being preincubated with BSA (1% in 0.01 M PBS; Sigma-Aldrich) for 1 h. Tissues were subsequently incubated in goat polyclonal antibody against Kit (R&D Systems, Minneapolis, MN; 1:500 in PBS, 0.01 M) at 4°C for 48 h. Immunoreactivity was detected with Alexa Fluor-488 donkey anti-goat IgG (Molecular Probes, Eugene, OR; 1:1,000, for 1 h at room temperature). Control samples were prepared in a similar manner, omitting primary or secondary antibodies from the incubation solution. All the antisera were diluted with 0.5% Triton X-100 in 0.01 M PBS (pH 7.2).
Tissues were examined with a Zeiss LSM 510 Meta confocal microscope (Zeiss) with an excitation wavelength appropriate for Alexa-488. Confocal micrographs are digital composites of Z-series scans of 10 to 20 optical sections through a depth of 10–20 μm. Images were reconstructed using Zeiss software, Adobe Photoshop 7.0 (Adobe, Mountain View, CA) and Corel Draw 7.0 (Corel, Ottawa, ON, Canada).
The ionic composition of the Krebs solution was as follows (in mM): 137.4 Na+, 5.9 K+, 2.5 Ca2+, 1.2 Mg2+, 15.5 HCO3−, 1.2 H2PO4−, 134 Cl−, and 11.5 glucose. The solutions were aerated with O2 containing 5% CO2, and the pH of the solutions was maintained at 7.2–7.3.
Drugs used were nifedipine, pinacidil, and sodium nitroprusside (SNP) (all from Sigma). Nifedipine and pinacidil were dissolved in DMSO to make stock solutions and were added to Krebs solution to make the desired concentrations, just before the use. SNP was dissolved in distilled water. The final concentration of the solvent in Krebs solution did not exceed 1/1,000. Addition of these chemicals to Krebs solution did not alter the pH of the solution.
Experimental values were expressed by the means ± SD. Statistical significance was tested using Student's t-test, and probabilities of less than 5% (P < 0.05) were considered significant.
When intracellular recordings were made from gastric circular muscle strips, an ongoing discharge of unitary potentials was observed as the most common activity (Fig. 1A). The resting membrane potential of cells generating unitary potentials ranged between −38 and −56 mV (mean −46.2 ± 4.2 mV; n = 79; n values represent the number of animals). The maximum amplitude of unitary potentials was ≤10 mV. Unitary potentials consisted of transient depolarizing and hyperpolarizing potentials. Occasionally (<1% of all cells impaled), STDs were also recorded (Fig. 1B). The resting membrane potential of cells generating STDs ranged between −40 and −56 mV (mean −48.3 ± 4.0 mV; n = 17, significantly larger than that of unitary potential-generating cells, P < 0.05). STDs were distinguishable from unitary potentials on the basis of the maximum amplitude (>20 mV) (Fig. 1C). STDs had highly variable amplitude (1–30 mV) and frequency (20–40/min). These properties were different from those of pacemaker potentials, which are generated rhythmically with the stable amplitude and frequency, recorded from ICC-MY of gastric antrum or small intestine (10, 19), and rather similar to those of STDs in smooth muscle of the urethra (9) or renal pelvis (24). Figure 1 shows examples of unitary potentials and STDs recorded from the same preparation.
Morphological features of cells generating unitary potentials and STDs.
To identify the morphology of the electrically active cells in the rat fundus, microelectrodes were filled with the fluorescent dye, propidium iodide, and hyperpolarizing current (intensity, 1 nA; duration, 100 ms; frequency, 3 Hz) was applied to inject the dye into impaled cells (19, 29). Discharge of unitary potentials came from spindle-shaped cells lying within circular layer with lengths 200–400 μm (Fig. 2Ab). These morphological features are identical to smooth muscle cells as has been previously reported in many studies of the GI tract (5, 8, 25). When propidium iodide was injected into the unitary potential-generating cells for sustained periods (>15 min), dye spread to neighboring smooth muscle cells in 9/14 preparations (Fig. 2Bb), demonstrating dye coupling between CSMC. In the remaining five preparations, propidium iodide also spread into another type of cell, which could be distinguished from smooth muscle cell by tapering ovoid nuclei and long, thin processes oriented along the axis of the CSMC fibers (Fig. 2Cb).
Using the same approach, STD-generating cells were visualized. STD-generating cells were bipolar with two primary processes emerging from a prominent perinuclear region (Fig. 3 Ab). These cells had the same morphological features with those labeled in Fig. 2Cb. These cell bodies had lengths of 18–25 μm and diameters at the widest point of 4–6 μm (n = 5). The length of these processes was 50–100 μm (n = 4). As in the case of unitary potential-generating cells, propidium iodide spread to neighboring cells after long injection periods (>15 min). Fig. 3Ab shows an example of the dye spreading from STD-generating cells to spindle-shaped, unitary potential-generating cells (CSMC) (n = 4). In one preparation, the dye spread from one bipolar cell to a second via long processes that connected the two cells together (Fig. 3Bb).
Kit immunohistochemistry was performed in an attempt to identify the type of cells filled with propidium iodide. In the rat gastric fundus, Kit-like immunoreactivity was localized to a population of spindle-shaped ICC-IM that possessed morphological features similar to the cell types that generated STDs (Fig. 4, A and B).
Unitary potentials recorded from circular muscle cells of rat gastric fundus.
The amplitude of unitary potentials differed between cells in the same preparation (Fig. 5A). Discharge of unitary potentials with amplitudes greater than 10 mV were observed in ∼30% of cells impaled (Fig. 5Ab). These results suggest that resolution of unitary potentials depends on the position and depth of a microelectrode in the preparation.
Because unitary potentials <1 mV were indistinguishable from baseline noise, unitary potentials with amplitudes >1 mV were sampled using amplitude/frequency histograms (Fig. 5C). With this cut off, the amplitude of unitary potentials ranged between 1 and 10.7 mV. The half-width (the duration of unitary potentials at the half-amplitude of the peak) of unitary potentials ranged between 42 and 408 ms. There was a correlation between the half-width of unitary potentials compared with the amplitude of unitary potentials (r = 0.65; Fig. 5D). These properties of unitary potentials were similar to those of unitary potentials recorded from single circular muscle bundles of the guinea pig gastric antrum (7) or from guinea pig gastric ICC-MY (10, 16).
Spontaneous transient depolarization recorded from ICC-IM.
Two types of STDs were detected in the rat fundus. In 12 preparations, bursts of STDs were generated with irregular intervals (burst-type STDs) (Fig. 6A). Among burst-type STDs, two preparations displayed discharge of rhythmic bursts of activity (Fig. 6B). The frequency of burst-type STDs ranged between 18 and 40/min (mean 27.1 ± 6.5/min; n = 12). On the other hand, single STDs with ∼20 mV in amplitude were also generated periodically (continuous-type STDs) in five preparations, exhibiting pacemaker-like activity (Fig. 8A). The frequency of continuous-type STDs ranged between 36 and 46/min (mean 42.2 ± 5.0/min; n = 5).
The variation of waveforms was seen in burst-type STDs. 1) Single-unit STDs summed to generate the diastolic slow depolarization, followed by rapidly rising large STDs (Fig. 6Ca). The shape of this type of STDs resembles regenerative potentials recorded from single circular muscle bundles of the guinea pig antrum (7). 2) The upstroke phase of STDs was developed with the continuous occurring of single-unit STDs, resulting in the large amplitude of STDs (Fig. 6Cb). 3) Summation of single-unit STDs did not reach the full-sized amplitude of STDs (Fig. 6Cc). 4) Single-unit STDs appeared during the repolarizing phase of summed STDs (Fig. 6Cd).
Figure 7A shows a distribution of burst-type STDs amplitude ranged from 1.3 to 31.9 mV. Although single-unit STDs were detected on both the upstroke and repolarizing phase of STDs (Fig. 6C), it was often difficult to measure the amplitude or half-width. Thus single-unit STDs with amplitudes >1 mV were used to make amplitude/frequency histograms. When the half-width of burst-type STDs was plotted as a function of burst-type STD amplitude, these two factors were positively related. The regression line calculated by using the least-squares method was well correlated (r = 0.82; see Fig. 7B).
The waveform of continuous-type STDs was also variable. There were at least four types of waveform in continuous-type STDs. 1) A rapid depolarization occurred abruptly in the upstroke phase of STDs (Fig. 8Ca). 2) A few of single-unit STDs were observed just before the generation of full-sized STDs (Fig. 8Cb). 3) A large step was found on the upstroke phase of STDs (Fig. 8Cc). 4) STDs were preceded by a slow depolarization (Fig. 8Cd). In addition, an incomplete depolarization, which fails to develop a full-sized STD, was observed (Fig. 8B).
To examine the amplitude/frequency histogram of continuous-type STDs, amplitudes >1 mV were measured. The amplitude of continuous-type STDs distributed in two groups (Fig. 9A), one with small amplitude ranging between 1.1 and 8.8 mV and another with large amplitude ranging between 12.5 and 26.5 mV. It was of interest to note that, in contrast to burst-type STDs, continuous-type STDs had few events that were 10 mV in amplitude. When the half-width of continuous-type STDs was plotted as a function of the amplitude, two groups were also observed; small amplitude with short half-width ranged between 30 and 98 ms (Fig. 9Ba), and large amplitude with long half-width ranged between 100 and 250 ms (Fig. 9Bb). Therefore these two groups were analyzed separately. However, there was no correlation between the half-width of continuous-type STDs compared with the small-sized amplitude of continuous-type STDs (r = 0.30; Fig. 9C). Also, there was no correlation between the half-width of continuous-type STDs compared with the large-sized amplitude of continuous-type STDs (r = 0.058; Fig. 9D). Unexpectedly, the half-width was relatively constant for continuous-type STDs with amplitudes raging between 12.5 and 26.5 mV. Thus it was likely that the small-amplitude group (Fig. 9Ba) and large-amplitude group (Fig. 9Bb) may represent single-unit STDs and summated STDs, respectively.
Summation of single-unit STDs in the gastric fundus.
Because the previous observations have demonstrated the possibility that single-unit STDs summated to generate larger-amplitude STDs as is the case for urethral STDs (9), the relationship between single-unit STDs and large-sized STDs was studied. In both burst-type (Fig. 10A) and continuous-type STDs (Fig. 10B), multiple steps were detected on the upstroke phase of STDs. Figure 10Bc shows a large step having an amplitude approximately two times larger than single-unit STDs (Fig. 10Ba). This may be due to simultaneous arising of two units in this STD.
Effects of membrane hyperpolarization on unitary potentials and STDs.
Pharmacological agents, such as NO donors and the KATP channel opener, which hyperpolarize GI smooth muscle tissues, are useful agents to distinguish passive electrotonic potentials from spontaneously active potentials. These agents have been shown to inhibit the amplitude of passive slow waves and increase the amplitude of active pacemaker potentials (17–20). Attempts were made to observe the effects of the NO donor, SNP, on the discharge of unitary potentials and STDs. SNP (3 μM) hyperpolarized the membrane of CSMC (10.0 ± 2.5 mV, n = 16) and abolished discharge of unitary potentials in nine preparations (Fig. 11A). However, in seven preparations, unitary potentials persisted in the presence of 3 μM SNP (data not shown). SNP (3 μM) also hyperpolarized the membrane of ICC-IM (11.1 ± 3.0 mV, n = 4) and increased the amplitude of STDs (Fig. 11Ca), without changing their half-width or dV/dtmax (Fig. 11C, b and c). SNP decreased the frequency of STDs (Fig. 11Cd). In the presence of SNP, only small-amplitude STDs were observed as is the case for unitary potentials recorded from BAPTA-loaded circular muscle preparations isolated from the guinea pig gastric antrum (7).
In the next series of experiments, the effects of the KATP channel opener, pinacidil, on unitary potentials and STDs were examined. Pinacidil (10 μM) hyperpolarized CSMC (20.4 ± 3.4 mV, n = 18), decreased the generation of discharge of unitary potentials in eight preparations (Fig. 12A), unchanged in four preparations, and increased in six preparations (data not shown). Pinacidil (10 μM) hyperpolarized the membrane of ICC-IM (18.8 ± 3.2 mV, n = 3) and increased the amplitudes and dV/dtmax of STDs (Fig. 12C, a and c), without changing their half-width or frequency (Fig. 12C, b and d).
Taken together, these results suggest that STDs recorded from ICC-IM in the rat gastric fundus are “active” membrane potential changes.
In the rat gastric fundus, two types of spontaneous electrical activities were observed. One was an ongoing discharge of unitary potentials (≤10 mV), similar to that seen in the mouse gastric fundus (1). The other was STDs. Combined with fluorescent dye injection and immunohistochemical studies with an antibody raised against Kit, it was found that discharges of unitary potentials were recorded from CSMC, whereas STDs occurred in ICC-IM. This is the first direct recording from fundic ICC-IM, demonstrating that these cells generate spontaneous electrical depolarizations that contribute to the overall excitability of this tissue.
Rat fundic ICC-IM exhibited two distinct types of STDs. In about 70% of ICC-IM, irregular bursts of noisy STDs were observed (burst-type STDs). Burst-type STDs were of variable waveforms, amplitudes, and half-widths. As shown in Fig. 1C, the small-amplitude unitary potentials recorded from CSMC may result from electrotonic spread of burst-type STDs. In fact, dye coupling exists between CSMC and ICC-IM (described below). In the remaining 30% of ICC-IM, periodic electrical activity was recorded (continuous-type STDs). This activity was a surprising finding because rhythmic membrane potential changes have not been reported previously in the gastric fundus. In contrast to burst-type STDs, continuous-type STDs were of relatively uniform amplitude. Given that two types of STDs are different especially in the waveforms and amplitude/frequency histograms, one may predict that they are activated by different mechanisms. However, it was found that, despite their differences, both types of STDs resulted from summation of single-unit STDs (Fig. 10), as in the case of urethral STDs (9). Hence the summation of single-unit STDs plays a fundamental role in the generation of large-amplitude STDs. The reason why two types of STDs were detected in the rat fundus is unclear. The number of ICC-IM varied from preparation to preparation because ICC-IM in the fundus are distributed among the smooth muscle cells without forming a distinct network (3, 23). Thus the simple explanation is that the activity of STDs depends on the number of ICC-IM present in the muscle preparations. Amplitude/frequency histograms of continuous-type STDs reveal that the activities of single-unit STDs are high enough to sum together easily to generate full-sized STDs constantly, suggesting that continuous-type STDs are generated in the muscle that has a large number of ICC-IM. On the other hand, large variations in the amplitude of burst-type STDs may result from variability in the probability of the summation of single-unit STDs produced by a small number of ICC-IM in fundic muscles. In addition, the difference of frequency between continuous-type STDs (high frequency) and burst-type STDs (low frequency with large variations) may be caused by a different number of ICC-IM contained in the preparations.
The ionic mechanisms underlying the generation of STDs in the rat fundus are uncertain. Previous studies have predicted that STDs are likely to be activated by the opening of Ca2+-activated Cl− channels (9, 32, 33). It has also been reported that Ca2+-activated Cl− channels may be involved in the generation of unitary potentials in the guinea pig gastric antrum (11). In contrast, recent studies suggested that Cl− conductances are unlikely to be related to unitary potentials in the mouse fundus, guinea pig fundus (1), and guinea pig proximal colon (12) because neither DIDS nor anthracene-9-carboxylate had any effect on the discharge of unitary potentials. In any case, both STDs and unitary potentials were reduced by agents that buffered [Ca2+]i, suggesting that [Ca2+]i-handling mechanisms are related to the generation of STDs/unitary potentials. Irrespective of the selectivity to Ca2+-activated Cl− channels of the agents known as Cl− channel blockers such as DIDS, the fact that those blockers had no effect on the discharge of unitary potentials in the fundus clearly demonstrates that ion channels related to the generation of the discharge of unitary potentials in the fundus are different from those in the antrum. DIDS inhibited the plateau component of pacemaker potentials recorded from ICC-MY in the guinea pig antrum (17). Hence the short duration of STDs may be due to the lack of DIDS sensitivity (Y. Kito, unpublished observations). Intense efforts should be required to reveal which types of ion channels are involved in the generation of STDs in gastric fundus ICC-IM.
Although dye coupling between smooth muscle cells and ICC-DMP or within ICC-DMP has been already shown (8), no evidence has yet been presented about dye coupling between smooth muscle cells and ICC-IM in the stomach. In the present study, the fluorescent dye propidium iodide diffused from CSMC to ICC-IM and vice versa, suggesting that they must be electrically connected with each other. These results support the previous study that the spontaneous electrical activities produced by ICC-IM appear to spread passively to CSMC to generate the discharge of unitary potentials in the fundus (1). As shown in Fig. 5A, the different amplitudes of unitary potentials were observed in the same preparation, indicating that the amplitude of the discharge of unitary potentials may depend on the coupling between CSMC and ICC-IM. However, unlike the good coupling between ICC-MY and CSMC in the antrum (4), the coupling between ICC-IM and CSMC seems to be poor because the amplitude of unitary potentials recorded from CSMC was still small (≤10 mV) (Fig. 2C). This may be due to a small number of connexins in the rat fundus, as is the case of the small number of connexin 43 in the mouse fundus compared with a greater number in the antrum (27). In this line, large-amplitude STDs generated in ICC-IM will give rise to attenuated, small passive waves of depolarization in an intact syncytium because fundic CSMC may not be well coupled to each other by gap junctions. Poor coupling between CSMC may be one of the reasons why the variable amplitudes of STDs were detected in the rat fundus. It should be noted that dye coupling was also observed among ICC-IM, suggesting that some of ICC-IM may be connected with each other by gap junctions to reinforce electrical coupling.
In the present study, membrane hyperpolarization induced by SNP or pinacidil increased the amplitude of STDs and decreased that of unitary potentials. Previous studies have demonstrated that the amplitude of active pacemaker potentials produced by ICC-MY was increased, whereas that of “passive” slow waves recorded from CSMC was decreased during application of SNP or pinacidil (17, 18, 20). In light of these observations, it seems reasonable to conclude that STDs are spontaneous active electrical activities and that the discharges of unitary potentials are passive electrotonic responses in the rat fundus. Because STDs were reduced and single-unit STDs were readily detected in the presence of SNP, as is the case of the effects of BAPTA-AM on unitary potentials recorded from single-bundle CSMC preparations of the guinea pig gastric antrum (7), SNP may reduce [Ca2+]i in the rat fundic ICC-IM. SNP seems to decrease the summation of single-unit STDs by inhibiting their generation, resulting in a reduction of the frequency of STDs. The mechanisms underlying how SNP causes membrane hyperpolarization were not addressed in the present study. It was proposed that SNP hyperpolarizes the membrane of CSMC indirectly via transduction mechanisms responsible for electrical responses localized in ICC-IM in the mouse fundus because SNP-induced hyperpolarizations were greatly attenuated in W/WV mouse fundus (2). In addition, a recent study demonstrated that cGMP elevated by SNP appears to cause release of Ca2+ from internal Ca2+ stores, which activates Ca2+-activated K+ channels, causing membrane hyperpolarization in the mouse fundic CSMC (15). Further studies are needed to elucidate the cellular mechanisms underlying SNP-induced hyperpolarizations in syncytial smooth muscle tissues.
In conclusion, unitary potentials and STDs were generated in CSMC and ICC-IM, respectively, in the rat fundus. ICC-IM exhibited two types of large-amplitude STDs, both of which were developed by the summation of single-unit STDs. Membrane hyperpolarization induced by pharmacological agents revealed that unitary potentials were passive electrotonic potentials, whereas STDs were spontaneously active potential changes. CSMC and ICC-IM seem to be connected by gap junctions because dye coupling was observed between these cell types. Taken together, it is likely that STDs generated in ICC-IM are the origin of unitary potentials recorded from CSMC in the rat fundus.
This project was supported by research grant from Japan Society for the Promotion of Science to Y. Kito (No. 19790460) and by NIH 57236 to S. Ward.
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