Digestive Diseases Division, Department of Medicine, Center for Ulcer Research and Education/Digestive Diseases Research Center and Brain Research Institute, University of California Los Angeles School of Medicine, Los Angeles, California 90095
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ABSTRACT |
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To prevent the blood-borne interference and reflex actions via neighboring organs and the central nervous system, the study was conducted in an in vitro isolated stomach-gastric vagus nerve preparation obtained from overnight-fasted, urethan-anesthetized rats. Afferent unit action potentials were recorded from the gastric branch of the vagus nerve. The left gastric artery was catheterized for intra-arterial injection. In vitro we found that 1) 55/70 gastric vagal afferents (GVAs) were polymodal, responding to CCK-8 and mechanical stimuli, 13 were mechanoreceptive, and 2 were CCK-responsive; 2) sequential or randomized intra-arterial injections of CCK-8 (0.1-200 pmol) dose-dependently increased firing rate and reached the peak rate at 100 pmol; 3) the action was suppressed by CCK-A (Devazepide) but not by CCK-B (L-365,260) receptor antagonist; 4) neither antagonist blocked the mechanosensitivity of GVA fibers. These results are consistent with corresponding in vivo well-documented findings. Histological data indicate that the layered structure of the stomach wall was preserved in vitro for 6-8 h. Based on these results, it seems reasonable to use the in vitro preparation for conducting a study that is usually difficult to be performed in vivo. For instance, because there was no blood supply in vitro, the composition of the interstitial fluid, i.e., the ambient nerve terminals, can be better controlled and influenced by intra-arterial injection of a defined solution. Here we report that acutely changing the ambient CCK level by a conditioning stimulus (a preceding intra-arterial injection of increasing doses of CCK-8) reduced the CCK sensitivity of GVA terminals to a subsequent test stimulus (a constant dose of CCK-8 intra-arterial injection).
vagus nerve; mechanoreceptive vagal afferent; cholecystokinin-responsive vagal afferent; cholecystokinin receptor antagonist
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INTRODUCTION |
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THE MAIN FEATURE of the digestive system is the diversity of anatomic structures of the gastrointestinal tract and associated glands and the complexity of related functions. In addition to the peripheral and central nervous system (CNS), the unique feature of the digestive system is the involvement of the enteric nervous system in the reflex control of gastrointestinal functions. For instance, an alteration in the mouth caused by food ingestion can initiate well-coordinated reflexes along the gastrointestinal tract that result in a series of physiological events (39), including the increase of intrarectal pressure (31). Therefore, when conducting an in vivo study on a gastrointestinal segment, it is important to be aware of the possible interference caused by unexpected reflex action.
It has been considered that, by monitoring unit action potentials traveling in cervical vagal afferent (CVA) fibers with an electrophysiological approach, the direct evidence of mechanosensitivity and CCK sensitivity of CVA terminals can be demonstrated more directly (15). In vivo gastric stretch- and tension-responsive CVA fibers have been investigated since the 1950s (18, 24), and the CCK responsivity of CVA terminals has also been studied for more than a decade (9). Subsequent studies indicate that gastric mechanoresponsive CVAs (including stretch and tension receptors) can be excited by 2-4 nmol/kg intravenous CCK-8 administration via the jugular vein from all 10 units tested (9). They can be activated also by intra-arterial injection close to the celiac artery via a catheter introduced from the carotid artery to the aorta (28), and the response was reported to be dose related (30). However, evidence of the inhibitory action of CCK on CVAs has also been reported. It was shown that intra-arterial injection of CCK-8 (100-200 pmol) in the ascending aorta close to the celiac artery (6) or intravenous injection via the jugular vein (50 pmol) reduced the activity of gastric-related CVAs, which closely followed the fall of intragastric pressure (15). Increasing the dose of CCK-8 from 50 to 500 pmol (iv) only prolonged the time course of the return to control levels (15). The inhibitory action of CCK was abolished by CCK-A receptor antagonist (Devazepide, 1.2 mg/kg iv) pretreatment (15). The excitatory action can be blocked by CCK-A (Devazepide) but not by CCK-B (L-365,260) receptor antagonist (30). Moreover, the response of CVAs to gastric load (2 ml) was not affected by CCK-A and -B receptor antagonist pretreatments (30). Results from our previous in vivo study also indicated that Devazepide (3.2 mg/kg iv) pretreatment blocked the activation of the ventral gastric branch of vagal afferent (GVA) discharge caused by 300 pmol intravenous CCK-8 (jugular vein) injection but did not affect gastric distension-induced GVA activation (40).
CCK was proposed to act directly at CCK-responsive CVA terminals (9, 29, 30). Because CCK receptors are widely distributed in the periphery, CCK may also act at neighboring organs, resulting in the alteration of gastric tension via the CNS or local reflex action (1, 10, 16, 26, 39). The tension change will, in turn, indirectly stimulate the mechanoreceptive CVA fiber (15). It is evident that the effect (excitatory vs. inhibitory) of exogenous CCK administration on gastric mechanoresponsive CVA fibers and the manner (direct vs. indirect) of the action of CCK are still subjects of considerable debate (6, 8-9, 15, 30).
The difference in animal species and in CCK dose has been considered to be the primary cause for these discrepancies (14, 15). However, the main and unique feature of the digestive system mentioned above should be considered as well. To prevent the blood-borne interference (17, 19) and reflex actions via neighboring organs (1, 10, 39) and the CNS (16, 26) and to gain better control over the local environment and easier access to gastric receptive fields, an isolated stomach-gastric vagus nerve in vitro preparation has been developed (35). In this preparation, the recording site of vagal afferent signals was moved from the cervical vagus to the gastric branch of the ventral subdiaphragmatic vagus nerve trunk (GVA). If the in vivo finding listed above can be reproduced in the in vitro preparation, it will indicate that reflex actions involving neighboring organs and the CNS are not essential for exogenous CCK to influence GVA fibers. Because the environment of the isolated stomach can be better manipulated, the research on the mechanism by which CCK activates GVA fibers can be conducted more easily.
Before the preparation can be employed extensively, it is necessary to provide evidence that indicates 1) during the time span of the experiment, GVA terminals should reproducibly respond to well-documented stimuli and generate well-defined unit action potentials; and 2) a typical layered structure of the stomach wall should be preserved after the isolated stomach was kept in the organ bath for 6-8 h. Moreover, a series of in vitro findings will be reported in this paper that includes the following: 1) based on the mechano- and CCK sensitivity, GVA fibers can be grouped into polymodal, mechanoreceptive, and chemoresponsive subgroups; 2) in a certain dose range, the excitatory action of CCK-8 was dose related; 3) evidence that the action was mediated by CCK-A but not by CCK-B receptor; 4) evidence that neither CCK-A or -B receptor antagonists blocked the mechanosensitivity of GVA fibers; and 5) histological evidence suggesting that the preparation can be well preserved throughout the time span of the experiment.
If the CCK action is indeed mediated by a receptor-binding mechanism, the existing ambient CCK level may affect the CCK sensitivity of GVA terminals. Because the blood-borne interference to the circulating CCK level (17, 19) and hence to the CCK level of interstitial fluid is difficult to be controlled in vivo, the appropriate answer to this extrapolation remains unknown.
Using the advantage of the in vitro approach, it becomes possible to alter the CCK concentration of interstitial fluid (the ambient CCK level of GVA terminals) via preinjection of a different dose of CCK-8 to the left gastric artery (conditioning stimulus), and the CCK sensitivity of CCK-responsive GVA terminals can be examined by a subsequent test stimulus (a constant dose of intra-arterial CCK-8 injection). Based on this protocol, we have been able to present evidence suggesting that acutely increasing the ambient CCK level in vitro may reduce the sensitivity of CCK-responsive GVA terminals.
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RESEARCH DESIGN AND METHODS |
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Animals
Sprague-Dawley male rats (250-300 g) were purchased from Harlan Laboratories (San Diego, CA) and were housed at the University of California, Los Angeles (UCLA), Division of Laboratory Animal Medicine. The room temperature was maintained at 20 ± 3°C, and the illumination was controlled in a 12-h light cycle beginning at 6:00 AM. Animals were fed with Purina Laboratory Chow (Ralston Purina, St. Louis, MO) and were provided with tap water ad libitum. Before the experiment, rats were fasted with free access to the water supply for 18-24 h. Before decapitation, animals were anesthetized with 25% of 1.5 g/kg urethan intramuscularly. Experimental procedures were approved by the Office for Protection of Research Subjects, Chancellor's Animal Research Committee UCLA.In Vitro Preparation
Experiments were conducted on an isolated rat stomach-gastric vagus nerve in vitro preparation, as previously described (35). To minimize the possible damaging effect of excessive residual blood on the isolated tissue, animals were exsanguinated by decapitation, and then a laparotomy was performed. Tissues from the lower-thorax esophagus to the proximal duodenum (~1 cm) and vagus nerves were isolated by a quick surgical operation. Within 5-10 min, the tissue was removed from the body and immersed in oxygenated modified Ringer solution containing 2 g/l D-glucose (perfusate; see Ref. 36). An opening was made at the nonglandular region of the stomach, through which the residual content was gently flushed. The tissue was then put in the main chamber of a Sylgard-coated (Dow Corning, Midland, MI) organ bath that was perfused continuously with oxygenated perfusate at 2.0-2.5 ml/min flow rate. Three partitions with a central hole divided the organ bath into the inlet and outlet chambers and the recording and main chambers. The perfusate was led into the inlet chamber by one of two pump heads (easy-load Masterflex model 7518-00) with Tygon tubing (6409-14, ID 1.6 mm) mounted on a Masterflex standard pump controller (Cole Parmer Instrument, Chicago, Il). The flowing solution was drained from the outlet chamber through another pump head with larger Tygon tubing (6409-16, ID 3.1 mm). These two chambers act as a buffer zone to minimize the disturbance caused by the flowing solution. The tissue was pinned in the main chamber with the ventral stomach facing up. The esophagus with attached vagus nerves was introduced in the recording chamber where a bipolar recording electrode was placed. For close intra-arterial injection, the left gastric artery was catheterized with polyethylene tubing (PE-10, OD = 0.61 mm) mounted to a 1-ml tuberculin syringe via a 27G1/2 precision Glide needle (9602 Becton-Dickinson, Rutherford, NJ). To reduce the metabolic rate and maintain the viability for a longer period of time, the bath temperature was set at 33 ± 1°C by a temperature controller (made by the electronic shop of the Department of Physiology, UCLA School of Medicine).Unit Action Potential Recording
In the recording chamber, the ventral gastric vagus nerve trunk was isolated from the surrounding connective tissue and placed on a miniholder. An opening (~2 mm long) was carefully made on the nerve trunk. With a pair of fine forceps (no. 5; A. Dumont & Fils), a thin nerve strand was cut and isolated from the opening. The distal cut end was placed on one lead of a bipolar recording electrode (platinum wire, 30 µm), and the other lead was connected to a slim connective tissue. Paraffin oil was used to prevent dehydration and short circuiting. The action potential of GVA fibers was sent to a preamplifier (DAM-6, ×100, 100-10 kHz band-pass filter; World Precision Instruments, Sarasota, FL). The signal was further amplified 300-750 times to give an action potential with a peak-peak amplitude of 1-5 volts, which was displayed on a digital storage oscilloscope (model 2211; Tektronix). An action potential responding to an adequate stimulus with a unique waveform and amplitude in an "all or none" manner was considered a unit action potential traveling along a single GVA fiber (see insets of Figs. 2A and 6B). In most cases, a thin nerve strand (~1/3 diameter of the recording electrode) may display three or more units with different amplitudes, although a single unit can be obtained in some cases.These unit action potentials, as raw data, were recorded on-line on a digital tape recorder (Sony high-density linear A/D D/A optical digital audio tape deck, DTC-700). They were simultaneously sent to a PC computer equipped with an A/D board (DT2831; Data Translation, Marlboro, MA).
Data Acquisition and Analysis
Using the acquisition module of WAVEFORM impulse analysis software (2), units within upper and lower threshold settings were acquired in the PC computer. Based on the amplitude and waveform, a particular unit can be traced off-line to match the waveform of the unit by the use of the analysis module of the software (2). The response pattern of different units can be analyzed further and displayed separately.The main response to intra-arterial CCK injection usually occurred within the first 5 min after a treatment, and the absolute pulse count per bin was related not only to the dose of CCK but also to the basal activity before treatment. Because the time span for each trial was 1 h long and the basal activity varied between units, the proper way to represent the response magnitude is to normalize these data by a quotient (Q, where Q = 5-min total spike count of posttreatment/that of pretreatment). Q >1 indicates an excitatory effect, Q< 1 indicates an inhibitory effect, and values close to 1 (±0.05) indicate no effect.
However, Q has its disadvantage; for instance, if the basal
ongoing activity is too low or zero for a silent unit, the denomination 0 and hence the Q
. This is the main cause
of dubious values. Based on our experience, for units with 5-min
average basal activity <0.1 pulse/s, Q cannot be used to
represent their response magnitude. Fortunately, these units only
occupy <10% (6/70) of total unit samples, and their response
magnitudes are represented separately by the mean of the 5-min firing
rate after the treatment (pulse/s). Data are presented as means ± SE. Statistical significance (P < 0.05) was assessed
with t-test or ANOVA for ordinary or repeated measurements
followed by the Tukey-Kramer or Dunnett's multiple comparison test.
Dubious values were evaluated using the filter of Grubbs criteria
(13).
Histological Study
In five experiments, at the end of the electrophysiological study, the isolated stomach was opened along the greater curvature and pinned flat on a Sylgard-coated petri dish containing oxygenated perfusate with the mucosa side facing up. The mucus was removed gently and rinsed with oxygenated saline. The saline was substituted by 4% paraformaldehyde to fix the specimen overnight. The fixed specimen was rinsed (3 × 10 min) with PBS, transferred to PBS containing 20% sucrose, and stored at 4°C overnight. The specimen was then embedded in optimum cutting temperature compound, cryostat-sectioned at 12-16 µm (Microm HM 500 OM), thaw-mounted on the slides, dried at room temperature, and stained with hematoxylin and eosin.The layered structure of the stomach wall was examined on an Olympus
IMT-2 inverted microscope equipped with ×4, ×10, ×20, and ×40
objectives and a paired off ×10 eyepiece. It had a light path
mounting port (MTU) for multitube attachment. A photo eyepiece (NFK
×3.3, LD) was inserted in the port via an adapter (MTV-3); a color
camera (model JE 3462RGB; Javelin Electronics) was mounted to the
microscope. The color image of the slide was displayed on a color video
monitor (Sony PVM-1343 MD) and was stored in a video cassette recorder
(Sony SVO-140) or captured by a video color printer (Sony CVP-G700) as
shown in Fig. 1. The ×600 image (Fig.
1E) was obtained by turning the magnification changer ring from ×1 to ×1.5 at ×40 objective observation (34).
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Drugs and Solutions
Modified Ringer solution was made as previously described (in mM: 140 NaCl, 5 KCl, 1 MgCl2 · 6 H2O, 1.3 Na2HPO4, 5 HEPES, 2 CaCl2 · 2H2O, and 10 D-glucose; pH 7.38 ± 0.02). All chemicals were purchased from Sigma (36). CCK fragment 26-33 (CCK-8 sulfated; Research Biochemicals International, Natick, MA) was dissolved in distilled water to make a 24 ng/µl (20 pmol/µl) stock solution. CCK-A (Devazepide) and CCK-B (L-365,260) receptor antagonists (Merck Sharpe and Dohme, St. Louis, MO) were dissolved in 50 µl of DMSO and Tween 80 to make a 1 µg/µl stock solution. Stock solutions were stored atExperimental Protocols
After the nerve strand was isolated and the initial search on mechanoreceptive fields of GVA fibers was completed, the whole experimental system was further monitored for ~30 min to ensure that a stable recording condition had been achieved. The amplitude and waveform of recorded unit action potentials can be affected by disturbances from unstable flow rate, fluctuating fluid level in the recording chamber, stretching the nerve strand due to not enough slack, and the interface between the nerve strand and electrode. After all possible sources of disturbances have been eliminated, the system usually can be stabilized within the first 30 min. When a tight contact between a good nerve strand and the electrode was ultimately formed, the reading of spike count per bin (1-min bin width) should be stable; otherwise, the nerve strand should be replaced by a new one.Response to injection of vehicle.
There was no blood supply in the in vitro preparation. A bolus of
intra-arterial vehicle injection was capable of transiently increasing
the intra-arterial pressure. To minimize a sudden change in pressure,
the time span for 0.1-ml intra-arterial injection was controlled at
10-15 s. The effect of four consecutive intra-arterial vehicle
injections on the basal activities of GVA fibers was studied in three
experiments. To make the procedure consistent with the experimental
trial, after each treatment, the catheter (with a dead space of 0.03 ml) was flushed with 0.1 ml vehicle. The interval between vehicle
tests was
15 min.
Response to intra-arterial CCK injection and mechanical stimulus. In 25 experiments, the response to CCK-8 and local mechanical stimuli was studied. To keep the preparation in a stable condition, the detailed search of mechanoreceptive fields was performed after the vehicle and CCK-8 (1 or 5 and 10 pmol) tests had been completed. The ventral surface of the stomach was divided into nine fields as shown in Fig. 3A. A brush made of camel's hair (2/0 Liquitex, 2/0 Kolinsky) moistened in the perfusate was gently rubbed over different fields to search for the most sensitive mechanoreceptive field. If there was no response to the gentle stimulus, an intermediate or a stronger push was exerted. A calibrated von Frey hair was used to locate the most sensitive mechanoreceptive spot inside the field. At the end of the experiment, 0.1 ml of 1% toluidine blue solution was injected intra-arterially to ensure that receptive fields were affected; if the fields were not affected, these data were excluded.
Dose-dependent activation by intra-arterial CCK-8 injection.
Six different doses of CCK-8 (0, 0.1, 1, 10, 100, and 200 pmol/0.1 ml)
were injected intra-arterially in either a sequential (4 experiments)
or a randomized (5 experiments) manner. After each treatment, the
catheter was flushed with an appropriate volume (>0.1 ml) of vehicle
to ensure that no residual solution was left. The time interval between
CCK tests was 30 min.
Effect of CCK receptor antagonists.
The effect of CCK receptor antagonist on the peak response (caused by
100 pmol intra-arterial CCK-8 injection) of GVA fibers was studied in
three experiments. Each GVA fiber was treated with consecutive
intra-arterial injections of vehicle, CCK-8 and L-365,260 (20 µg),
CCK-8 and Devazepide (20 µg), and CCK-8. The time interval after
vehicle to the next treatment was 15 min, the interval after CCK-8
was
30 min, and the interval after receptor antagonist was ~15 min.
In two separate experiments, the effects of Devazepide (20 µg) on the
response of GVA fibers to 200 pmol CCK-8 (the highest dose) and of
L-365,260 (20 µg) to 10 pmol CCK-8 (a lower dose) were studied.
Effect of an acute change in ambient CCK level. In five experiments, three different ambient CCK levels were mimicked acutely by intra-arterial injections of 0.1 or 1.0 and 10 pmol CCK-8 (conditioning stimulus) at 5-10 min before the test stimulus. The effect of an acute alteration in ambient CCK levels on the sensitivity of CCK-responsive GVA terminals was examined by a test stimulus (intra-arterial injection of 5 pmol CCK-8).
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RESULTS |
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Time Span of the Experiment and Viability of the In Vitro Preparation
The time span of an experiment is the time period from start to stop of data acquisition. The average time span of 50 experiments reported in this paper was 3.32 ± 0.19 h (range 1.46-7.58 h). Unit action potentials (Figs. 2A and 6B, insets) were monitored continuously throughout the experiment to ensure the preparation was viable. The result of histological study on five specimens from five experiments indicates that the layered structure of the stomach wall had no sign of deterioration (Fig. 1). This is consistent with the electrophysiological finding.
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Basal Activity
The 5-min average firing rate, just before the first intra-arterial vehicle injection, was used to represent the basal activity. A total of 70 single GVA fibers was analyzed from 25 experiments. The basal activity ranged from 0 to 4.23 pulses/s. Among them, 64 were units with high-rate basal activity (Response to Intra-Arterial Vehicle Injection
Figure 2A shows original records of a GVA fiber that responded to four intra-arterial vehicle (0.1 ml) injections with respective Q values of 1.04, 1.15, 0.73, and 1.05. Although there was variation between different units and different trials of the same unit, the overall difference of 11 units from 3 experiments (Fig. 2B) was statistically insignificant.Responding to CCK and/or Mechanical Stimuli
Of 70 GVA fibers, 55 are polymodal, responding to both mechanical and CCK stimuli. Eighty percent of the most sensitive mechanoreceptive field (Fig. 3B) was located in areas 1, 2, 4, and 5 (Fig. 3A). Fifty of 55 units with high-rate basal activity (1.20 ± 0.12 pulses/s) responded to intra-arterial injection of 1, 5, and 10 pmol CCK-8 (Fig. 3C, bars 2-4). Fifty of 55 units with high-rate basal activity (1.20 ± 0.12 pulses/s) responded to intra-arterial injection of 1, 5, and 10 pmol CCK-8 (Fig. 3C, bars 2-4). The asterisks indicate that the Q value is significantly higher than that of vehicle. One silent unit did not respond to vehicle but was activated by 1 pmol CCK-8 with a firing rate of 0.25 pulse/s.
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Thirteen of seventy units were mechanoresponsive GVA fibers that did not respond to or were slightly inhibited by 1-10 pmol intra-arterial CCK-8 injection but were activated by local mechanical stimulus. More than 90% of the most sensitive mechanoreceptive fields were located at areas similar to those of polymodal fibers (Fig. 3B). Twelve of thirteen units showed a high rate of basal activity (1.51 ± 0.33 pulses/s) that responded to intra-arterial vehicle injection with a significantly higher Q (1.55 ± 0.29) than that (1.14 ± 0.05) of polymodal units (2-tailed P < 0.05, unpaired t-test). This result suggests that mechanoreceptive units are more sensitive to intra-arterial pressure changes. Although the Q in response to intra-arterial CCK-8 was lower than that in response to vehicle, the difference was not significant (2-tailed P > 0.05, paired t-test). Only one unit with a low rate of basal activity (0.03 pulses/s) responded to vehicle and intra-arterial CCK-8 injection, with respective 5-min average firing rates of 0.19 and 0.04 pulses/s.
Two CCK-responsive units, with basal activities of 0.22 and 1.32 pulses/s, responded to intra-arterial CCK-8 injection, but no gastric mechanoreceptive fields could be located.
Dose-Related Response to Intra-Arterial CCK-8 Injection
Figure 4A shows the response of a GVA fiber to six consecutive intra-arterial injections of CCK-8 at respective doses of 0, 0.1, 1, 10, 100, and 200 pmol. Although the detailed response pattern of each unit may be different, this unit dose dependently responded to 0.1-200 pmol CCK-8 with Q values of 1.50, 2.78, 7.56, 15.06, and 18.32 (Fig. 4A, 2-6). When the dose was increased from 1 to 100 pmol, the latency for reaching the initial rising phase of spike count was reduced from ~45 to ~15 s. However, there were no clear latency changes when the dose was raised from 0.1 to 1.0 and from 100 to 200 pmol.
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To determine whether the dose-related response was the result of an accumulative action of CCK sequential administration, an additional five experiments were performed. Although the left gastric artery was injected with the same range but with randomized doses of CCK-8, the same dose-related response and latency reduction were seen. Figure 4B presents the response pattern of 12 units to randomized doses of intra-arterial CCK-8 injections. The response starts from 0.1 pmol and reaches the peak at 100 pmol. Ten units (from 4 additional experiments) show the same dose-dependent pattern as Fig. 4B in response to sequential CCK-8 administration (data not shown). These results indicate that accumulative action is not the main cause of dose-related changes in Q and latency.
Effect of CCK-A and CCK-B Receptor Antagonists
Figure 5A shows that CCK-A (Devazepide, bar 5) but not CCK-B (L-365,260, bar 3) receptor antagonist blocks the peak response (100 pmol CCK-8) of GVA fibers (bars 2, 4, and 6). Figure 5, B and C, shows results from two separate experiments that further indicate that Devazepide also blocked the response of GVA fibers to the highest dose (200 pmol) of CCK-8 activation; however, L-365,260 could not significantly suppress the response of GVA fibers, even with 10 pmol (a lower dose) intra-arterial CCK-8 injection.
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Effect of an Acute Change in Ambient CCK Level
GVA fibers reproducibly responded to three consecutive test stimuli (intra-arterial injections of 5 pmol CCK-8; Fig. 6A, bars 2-4). The asterisk indicates that the Q was significantly different from that caused by intra-arterial injection of vehicle. Although there was variation, differences among units and between CCK trials of the same unit were not significant.
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Figure 6B shows that a GVA fiber was not activated by intra-arterial injection of vehicle (Fig. 6B, 1) but dose dependently responded to three conditioning intra-arterial CCK-8 injections with increasing doses from 0.1 pmol (Fig. 6B, 2, left) to 1.0 (Fig. 6B, 3) and 10 (Fig. 6B, 4) pmol. Five to ten minutes later, the CCK sensitivity of the unit was examined by a test stimulus, a constant dose of 5 pmol intra-arterial CCK-8 injection. Note that when conditioning stimulus and hence ambient CCK levels were increased from 0.1 to 1 and 10 pmol caused by the conditioning stimulus, the Q to the subsequent test stimulus decreased from 2.29 to 1.54 and 1.18 (Fig. 6B, 2-4, right), respectively. Figure 6C shows data for 15 units. Although there was variation among the response patterns of different units, the result showed the trend, i.e., when ambient CCK level was acutely raised, the CCK responsiveness of GVA fibers was correspondingly decreased. The data suggest that the terminal's CCK sensitivity shows a negative correlation to the amount of CCK to which they were exposed.
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DISCUSSION |
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In vitro, we have been able to identify CCK and/or mechanoresponsive GVA fibers. Because data were obtained from an isolated organ, the possible involvement of the central action on gastric function via long vago-vagal reflex (1, 26) was eliminated. The peripheral action through intersegmental reflexes, such as esophagogastric and duodenogastric reflexes (10, 39) and the effect of respiratory movement on the basal activity of GVA fibers (9) was also minimized or avoided. Because these neuronal signals were intercepted from the ventral GVA, the sample bias was better controlled than that recorded from the cervical vagus, and the accessibility of receptive fields was also significantly improved. Although these advantages may benefit the current study and may help to resolve the disadvantage of the in vivo approach, we should be aware of the possible effect of shortcomings inherited from the in vitro approach. For instance, in vitro 1) there was no blood supply, and 2) the connection of GVA fibers with their soma in the nodose ganglion had been interrupted. Therefore, before the in vitro isolated stomach-vagus nerve preparation can be used in our future studies, the following critical questions should be addressed. 1) How long can a viable organ preparation be kept in vitro? 2) Are these unit action potentials recorded from the in vitro preparation compatible with those obtained from the in vivo study? 3) Is the result of the current study comparable with that of the in vivo well-documented study? 4) What is the significance of current new findings?
How Long Can a Viable Organ Preparation be Kept In Vitro?
An isolated stomach can be kept viable in vitro for >7 h. The average time span of 50 experiments included in this study was 3.32 ± 0.19 h. Actually, the termination of each experiment was due to the time limit of experimental protocols. For 50 experiments, the time span for the longest protocol was >7 h, whereas the shortest time span was ~2 h. At the end of each experiment, the responsiveness of the GVA terminals and the appearance of unit action potentials indicated that the preparation was viable and the terminal of GVA fibers was functional. Therefore, the experiment could have been carried out further, if necessary.All of our in vitro experiments were terminated on purpose by intra-arterial injection of 0.1 ml of 1% toluidine blue. The entire staining area encompassed the receptive field of GVA fibers to be tested. Most units responded to the intra-arterial dye injection with initial high-frequency discharge and then gradually reduced or even stopped firing. This procedure was very helpful to ensure that the injected solution was delivered to the interstitial fluid where GVA terminals were located. Based on the evidence mentioned above, it seems justifiable to believe that, at the end of experiment, these GVA terminals were responding, and the intra-arterial injection was valid.
Although we did not pursue the actual entire survival time of the in vitro preparation, it is reasonable to estimate that, with our setup, the viability of the preparation can be maintained in vitro for >7 h. Because most of our experiments were terminated voluntarily at the 3rd hour, the validity of these data was secured. The histological evidence from five experiments, as presented in Fig. 1, sustains the above notion.
Are These Unit Action Potentials Recorded From the In Vitro Preparation Compatible With Those Obtained From the In Vivo Study?
The unit action potential is compatible with that recorded in vivo. In responding to an adequate stimulus, unit action potentials with a unique waveform can be recorded reproducibly from a GVA fiber in an all-or-none manner. Once the stimulus reached the threshold, a full-fledged action potential was produced. Further increase in the intensity produced no increment or other change so long as experimental conditions remained the same (18, 24). As shown in Figs. 2A and 6B, insets, all unit activities of GVA fibers reported in this paper meet these criteria and show a positive (upward)-negative (downward) biphasic waveform with smooth rising and falling phases, indicating normal unit action potentials. This result is consistent with our previous findings from in vivo studies on unit action potentials of unmyelinated muscle afferent fibers and slowly conducting spinal white matter nerve fibers (37, 38). Unit action potential recorded from myelinated fibers displays a high-amplitude, short-duration (<1 ms) potential with a monophasic (positive) waveform. Therefore, on the audio monitor, a louder, high-frequency sound can be easily distinguished from a sound generated by unmyelinated fibers.Although we did not measure the conduction velocity of units to be studied, because they all demonstrated a biphasic unit action potential with low-amplitude, long-duration (>2 ms) potential and lower frequency sound, it is reasonable to believe that these GVA fibers were unmyelinated. These findings are consistent with the results from the morphological study reported by Prechtl and Powley (25), i.e., >99% of ventral GVA fibers are unmyelinated. Based on the data presented, we are convinced that our setup can keep the electrophysiological property of GVA terminals for >7 h without any obvious signs of deterioration.
These results suggest that, during the first 6-8 h after being separated from the cell bodies and being kept in vitro, the electrophysiological properties of GVA fibers and their terminals show no sign of alteration. This notion is consistent with results from our previous studies on three different in vitro preparations. These preparations were 1) the splanchnic afferent-mesentery in vitro preparation in which thin myelinated and unmyelinated fibers were recorded (2); 2) the esophagus-gastric vagus nerve in vitro preparation in which unmyelinated fiber was investigated (36); and 3) the saphenous nerve-skin patch in vitro preparation, which was used to investigate mainly myelinated fibers (33).
Is the Result of the Current Study Comparable With That of the In Vivo Well-Documented Study?
Results of the current study are comparable with those of well-documented in vivo studies. Most of studies on the unit activity of vagal afferent fibers were conducted in vivo at the CVA (3, 6-9, 15, 16, 18, 20, 24, 28-30). Two exceptions include the in vivo study on the mass discharge of the gastric vagus nerve trunk (23) and on unit activities of ventral gastric vagus nerve strands (40).Study I. Explicit criteria for classification of gastrointestinal tract-related CVA fibers have not been agreed upon (14). In vitro, we found that, at the ventral gastric vagus, most of the GVA fibers (>78%) were polymodal, responding to exogenous CCK intra-arterial injection and local mechanical stimulus with increasing firing rate. With the use of a dissecting microscope, even the most sensitive gastric mechanoreceptive field of these units can be located. These findings are consistent with results from in vivo studies on the CVA reported by other investigators (9, 29) and our previous study on the ventral gastric vagus nerve strand (40).
About 18% of GVAs are mechanoreceptive. Although the distribution of mechanoreceptive fields was similar, the excitatory sensitivity to a transient elevation of intra-arterial pressure was significantly higher than that of polymodal fibers. Moreover, instead of an enhancement effect, intra-arterial CCK-8 injection tended to suppress the activity of mechanoreceptive GVAs, although the difference from the vehicle trial was statistically insignificant. This finding may account for the results of in vivo studies reported by other investigators on the inhibitory effect of exogenous CCK on the activity of preloaded gastric mechanoresponsive CVAs (6, 15). Only ~3% of GVAs were CCK-responsive fibers. Because they did not respond to local mechanical stimulus, no gastric mechanoreceptive fields could be located. It is possible that the threshold was so high that the local mechanical stimulus we used was not strong enough to activate the terminal of these GVA fibers.Study II. If the action of CCK-8 is indeed mediated by a receptor-binding mechanism, the response of GVA terminals to close intra-arterial CCK-8 injection should be dose related. In vivo, the dose-related action, both excitatory and inhibitory, of exogenous CCK on the gastric CVA has been reported (9, 15, 30). In the current study, we provided more systematic and stronger data indicating that, in vitro, GVA fibers responded to intra-arterial CCK-8 injection with increasing firing rate in a dose-dependent manner. These excitatory responses were not caused by the accumulative action of CCK. As presented in Fig. 4, these data show that either sequential or randomized intra-arterial injection of CCK-8 (0.1-200 pmol) in GVA terminals did display dose-dependent excitatory responses; therefore, the results from our in vitro study are consistent with the notion generally accepted.
Study III. The results from the current in vitro study also confirm the findings of our previous in vivo study that indicate that CCK-A receptor antagonist blocked the excitatory action of CCK-8 on GVAs but did not alter the mechanoresponsiveness of GVA fibers (40). This finding is consistent with results from reported morphological, functional, and electrophysiological in vivo studies (4, 12, 21, 30).
In the current study, we have also demonstrated that the CCK-B receptor antagonist L-365,260 did not block a lower dose (10 pmol) of CCK-8 action on GVA terminals. This result suggests the lack of CCK-B receptor expression at GVA terminals. However, results from recent autoradiography studies reported by Moriarty et al. (22) indicate that both CCK-A and CCK-B receptors are synthesized by nodose ganglion cells, and receptor proteins are transported to the periphery along afferent fibers. However, the details of the destination were not specified. It is possible that type B receptor protein-containing vagal afferent fibers do not terminate at the stomach. This notion is supported by the result of a recent immunohistochemistry study (32). On the basis of these comparisons, it seems reasonable to believe that it will be beneficial to use the in vitro preparation as an additional tool in the study of vagal afferent signaling.New Findings and Significance
Finding 1. The most significant finding of our current study is that we have been able to provide evidence to show the effect of acute alteration in ambient CCK level on the CCK sensitivity of GVA terminals. If the involvement of receptor-binding mechanisms is correct, the existing ambient CCK level may have influenced the CCK sensitivity of GVA terminals. Although this is a reasonable extrapolation, testing this working hypothesis is difficult in vivo. This is because in vivo, there is blood-borne interference, and there is no easy way to control the ambient CCK level. However, with the use the in vitro isolated stomach preparation, this difficulty can be overcome. As mentioned above, in vitro, because there is no circulating blood supply, the ambient CCK level can be mimicked by intra-arterial injection of different doses of CCK-8 (conditioning stimulus). Five to ten minutes later, the CCK sensitivity of GVA terminals can be examined by a test stimulus (5 pmol CCK-8 ia injection). Using this experimental protocol, we have been able to demonstrate that acutely elevating the ambient CCK level results in the reduction of CCK sensitivity of GVA terminals. This finding further upholds the receptor-mediated hypothesis.
Finding 2. Several laboratories have reported that CCK may influence tension receptors of the upper gastrointestinal tract, including the duodenum and stomach of different animals, such as rats (7, 9, 29), sheep (8), and ferrets (3, 6). However, there is still debate about whether the action is directly aimed at the terminal of vagal afferents (9, 29) or on the gastric smooth muscle and whether the change is in muscle tension indirectly, causing the excitatory or inhibitory influence on the GVA terminal (6, 8, 15). Recent evidence indicates that CCK-A and -B receptors were distributed in guinea pig gastric muscle (5, 11), whereas in rats only CCK-B was distributed (27). In rats, if the action of CCK-8 on GVA fibers is an indirect effect caused by alteration of gastric muscle tension, the action should be blocked by application of CCK-B receptor antagonist.
Because CCK-B receptors are widely distributed, it is difficult to ensure that CCK-B receptor antagonist is delivered only to the stomach and acts in a restricted way on the gastric smooth muscle. Therefore, in vivo it is difficult to examine this notion. By contrast, because the experimental environment can be better controlled, the study can be conducted in the in vitro isolated stomach preparation. Our data indicate that the activation of GVA fibers by intra-arterial CCK-8 injection was abolished by CCK-A but not by CCK-B receptor antagonist.Finding 3. The debate on the mechanism of action of CCK also introduces another controversy that was centered on whether there is a polymodal GVA fiber responding to both mechanical stimulus and intra-arterial CCK injection (18). The morphological evidence reported by Berthoud and Powley (4) shows that, in rats, a single GVA fiber innervating the fundic region had separate collateral branches innervating both the smooth muscle layer and the myenteric ganglia. Collateral terminals in the muscle layer and myenteric ganglia form different types of endings. They speculate that their finding may be the morphological substrate for the polymodal nature of GVA fibers. Our data demonstrate that, of 70 single GVA fibers, >78% (55/70) are polymodal, responding to both intra-arterial CCK-8 injection and local mechanical stimuli. Our findings provide electrophysiological evidence to support the notion that, in the rat, most of the GVA fibers are polymodal in nature.
Finding 4. The mechanoreceptive field of GVA fibers was searched for with a fine camel-hair brush (tip <2 mm); the brush was pressed over nine different fields of the ventral surface of the stomach (Fig. 3A). GVA fibers can be activated by a mechanical stimulus applied over the center and the off center (in lesser degree) of the mechanoreceptive field. As reported by other investigators (9, 29) with a dissecting microscope, the most sensitive spots can be located as well. In vitro, the most sensitive field or spot of 82% of 68 unit samples, including polymodal and mechanoreceptive units, had been located in the proximal half of the fundus and fundic-corpus borderline, i.e., Fig. 3A, fields 1, 2, 4, and 5. The other 13% were located at the center of the corpus, central to the antrum and proximal duodenum (fields 7, 8, and 9), and only 5% were at the greater curvature side of the fundus (fields 3 and 6), which is consistent with the reported morphological findings (4). These results suggest that GVA fibers were not distributed evenly. Results from an in vivo study reported by Davison and Clarke (9) indicate that 60 unit samples were isolated from all regions of the stomach, but there was an apparent greater density of antrum-pyloric receptors than lower esophageal sphincter-cardiofundic receptors (42 vs. 19 units). Although the reason for this discrepancy is currently unknown, one possibility is that the stomach environment in vivo is different from the in vitro environment. Also, their study was conducted at the cervical vagus, whereas our study was conducted at the gastric branch of the subdiaphragmatic vagus nerve.
Data presented in this report evidently indicate that GVA fibers can be studied in an isolated stomach-gastric vagus nerve in vitro preparation. Because the experimental condition can be controlled better in vitro, some experimental protocols, which are difficult to perform in the in vivo preparation, can be conducted in the in vitro setup. Most of the data presented are comparable to corresponding in vivo data reported by other investigators or to results of our previous study, although some discrepancies exist. These results warrant the approval of using this preparation in future studies. ![]() |
ACKNOWLEDGEMENTS |
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We thank Dr. Y. Tache for helpful suggestions on this manuscript, Dr. D. W. Adelson for assistance in designing methods of computer problem solving, and Drs. P. Q. Yuan and M. Miampamba for help in the histological study.
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FOOTNOTES |
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This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48476.
Address for reprint requests and other correspondence: J. Y. Wei, Dept. of Medicine, UCLA School of Medicine, 900 Veteran Ave., Rm. 14-129, Los Angeles, CA 90095-1786.
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.
Received 7 September 1999; accepted in final form 12 April 2000.
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