Initiation of distension-induced descending peristaltic reflex in opossum esophagus: role of muscle contractility

A. Muinuddin and W. G. Paterson

Gastrointestinal Diseases Research Unit and Departments of Medicine and Physiology, Queen's University, Kingston, Ontario, Canada K7L 5G2


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The balloon distension (BD)-induced descending peristaltic reflex in the opossum smooth muscle esophagus is abolished in vitro when a Ca2+-free Krebs solution is placed at the site of distension, suggesting that either synaptic transmission occurs at the origin of the reflex or initiation of the reflex requires the development of muscle tension in response to BD. To test the latter possibility, an 8- to 10-cm length of smooth muscle esophagus was placed in a dual-chamber organ bath, isolating the stimulating (orad) from the recording site (aborad). Nifedipine addition to the orad chamber (i.e., site of distension) inhibited the BD-induced "off" contractions in both chambers in a concentration-dependent manner. However, the aborad response to electrical field stimulation (EFS) was unaffected. Atropine addition to the orad chamber had no effect on BD or EFS responses in either chamber. To examine the effects of these agents on tonic contractility, an isobaric barostat was employed. Pressure-volume curves were not altered by Ca2+-free Krebs solution, nifedipine, or TTX, suggesting that resting esophageal tone is not dependent on neural factors or muscle contractility. However, both Ca2+-free Krebs solution and nifedipine markedly decreased phasic contractions over the top of the distending bag. These observations suggest that local, stretch-induced phasic muscle contraction is required for initiation of the BD-induced descending peristaltic reflex.

secondary peristalsis; smooth muscle; tone; calcium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE DISTENSION-INDUCED DESCENDING peristaltic reflex in the smooth muscle esophagus is a highly complex motor activity, which is influenced by central and peripheral levels of control as well as by myogenic properties of the musculature (7). Similar to the small intestine, the esophageal response to intraluminal distension consists of proximal excitation and distal inhibition, which serves to ensure aborad propulsion of the intraluminal bolus (15). Although this reflex has been extensively studied in the small intestine (1, 5, 17), little is known about the intramural mechanisms responsible for the initiation and propagation of this reflex in the smooth muscle esophagus.

Studies from our (16) laboratory using a triple-chamber organ bath that permitted different sections of the smooth muscle esophagus to be chemically isolated, while keeping the neuromuscular apparatus intact, helped characterize the intramural neural pathways involved in the esophageal distension-induced descending peristaltic reflex. Balloon distension (BD) in the orad chamber evoked membrane hyperpolarization in the aborad chamber, which was followed, on balloon deflation, by depolarization, spike burst, and circular smooth muscle contraction. Addition of TTX to any of the chambers abolished this reflex, indicating its neurogenic nature. Furthermore, synaptic blockade using 0 Ca2+-20 mM Mg2+ Krebs solution abolished the BD-induced aborad "off" contractions when placed in either the aborad or orad chamber, but not when placed in the intermediate chamber, indicating that long descending intramural neurons mediate the BD-induced descending peristaltic reflex in the opossum esophagus. The nitric oxide (NO) synthase inhibitor nitro-L-arginine methyl ester, when placed in the aborad chamber, abolished this reflex, indicating that NO is the final mediator. Though 0 Ca2+-20 mM Mg2+ Krebs solution significantly attenuated the reflex (suggesting that synaptic transmission occurs at the origin of the reflex), none of the putative neurotransmitter antagonists tested were able to significantly alter the response. This included addition to the orad chamber of antagonists of the sensory neurotransmitters substance P and calcitonin gene-related peptide (CGRP). One explanation for these observations is that a synapse does exist between a distension-sensitive mechanoreceptor and the long descending intramural neurons, although it involves a neurotransmitter not yet tested for. However, stimulation of sensory neurons with capsaicin or addition of the putative sensory neurotransmitter substance P or CGRP to the orad chamber failed to elicit the descending peristaltic reflex in this preparation (16). Another possibility is that the inhibitory effect of 0 Ca2+-20 mM Mg2+ Krebs solution in the orad chamber is due to decreased muscle tension development in response to stretch. In other words, the distension-induced descending peristaltic reflex may be initiated by muscle contraction in response to stretch, rather than by the stretch itself. Thus the aim of this study was to test the hypothesis that local smooth muscle contractility in response to BD is necessary to initiate the distension-induced descending peristaltic reflex.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

All procedures were approved by the Queen's University Animal Care Committee. Opossums (Didelphis virginiana) of either sex, weighing between 1.4 and 6.0 kg, were anesthetized with a tail vein injection of 40 mg/kg body wt of pentobarbital sodium (Somnotol; MTC Pharmaceuticals, Canada Packers, Cambridge, ON, Canada). The thoracic cavity and abdomen were opened along the midline, and the length of esophagus required was measured in situ, following which the entire smooth muscle esophagus and part of the stomach were removed. The animal was euthanized with an intracardiac injection of pentobarbital sodium. The esophagus was then immediately placed in a dissecting dish containing modified Krebs solution. The composition of the Krebs solution was as follows (in mM): 115 NaCl, 4.75 KCl, 1 NaH2PO4, 1.2 MgSO4, 25 NaHCO3, 2.5 CaCl2, and 11 glucose. The Krebs solution was constantly bubbled with a gas mixture of 5% CO2-95% O2 and maintained at 34°C (i.e., the physiological core body temperature of the opossum) with pH between 7.2 and 7.4.

Descending Peristaltic Reflex: Double-Chambered Bath Experiments

The double-chamber bath used was a modified version of that described by Paterson and Indrakrishnan (16). The setup consisted of a 10-ml orad chamber and a 150-ml aborad chamber that were separated by a Plexiglas divider containing an 8-mm-diameter hole (Fig. 1). The distal 8- to 10-cm length of opossum smooth muscle esophagus was threaded horizontally through the hole in the Plexiglas divider, and inert silicone grease (Dow Corning, Midland, MI) was applied around the esophagus-Plexiglas interface to create a seal between chambers. The effectiveness of this seal was tested by adding trypan blue solution (Sigma Chemical, St. Louis, MO) to the orad chamber and visually checking for leakage of this dye into the aborad chamber. Experiments only proceeded if no leakage of dye was observed. Previous studies in our (16) laboratory also employed functional tests to confirm the effectiveness of these seals. In these studies, addition of TTX (10-6 M) to one chamber did not affect nerve-mediated responses evoked in the adjacent chamber.


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Fig. 1.   Schematic representation of the double-chamber organ bath used to study the descending peristaltic reflex. Both transient intraluminal balloon distension (BD) (~5 s) and electrical field stimulation (EFS) (3-s train, 1-ms pulse duration, 10 Hz, 60 V) were performed in the orad chamber. Esophageal contractile activity was recorded from both the orad and aborad chambers with a minimally perfused manometry catheter (0.15 ml/min).

Intraluminal manometry was performed using a catheter system that consisted of a distending balloon, and five polyvinyl recording tubes (0.8-mm inner diameter, 1.16-mm outer diameter) that were glued together with tetrahydrofuran (BDH Ontario). A 1-mm diameter side-hole orifice was cut in each of four recording tubes, and the tube distal to the orifice was sealed, so that pressure recording ports were created at sites 1, 2, 3, and 4 cm from the distal end of the balloon. The fifth tube was used to inflate and deflate the balloon. During pressure recording, each catheter lumen was minimally perfused with distilled water at a rate of 0.15 ml/min using a pneumohydraulic capillary infusion system (model PIP-3, Mui Enterprises, Mississauga, ON, Canada). When secured in the chambered organ bath, intraluminal pressures were recorded at a site 1 cm distal to the balloon (i.e., orad chamber) and at sites 2, 3, and 4 cm distal to the balloon (i.e., aborad chamber). The catheters were connected to pressure transducers (Transpack II; Sorenson Research, Abbott, Salt Lake City, UT). All data were collected by the WinDaq/200 data acquisition system (Dataq Instruments, Akron, OH) and stored on hard disk. A latex balloon, 2 cm in length and made from a finger cot (size medium 28030, Realmont, Cowansville, QC, Canada), was placed over the top of the catheter and then folded back on itself. The ends were sealed with silk suture and Vetbond tissue adhesive (no. 1469, 3M, St. Paul, MN). With this setup, the balloon produced circumferential distension of the esophagus around the manometry tube when inflated with air via a syringe.

Experiments proceeded only if the contraction amplitude of the control responses was >10 mmHg in both the orad and aborad chamber. The protocol consisted of a 20- to 25-min stabilization period followed by baseline control responses for BD (2 ml vol · 5 s) and/or electrical field stimulation (EFS; 3-s trains of square wave pulses of 10 Hz frequency, 1-ms pulse duration, and 60-V stimulus strength applied to the adventitial surface of the esophageal segment via a bipolar silver chloride electrode and Grass S88 stimulator). Each stimulus was performed at least three times, with at least 3 min allowed to elapse between each stimuli. Any response beginning after the termination of the stimulus was defined as an off contraction. In one series of experiments, cumulative concentration-response curves were established for the effect of nifedipine (10-8 to 10-4 M applied to the orad chamber) on off contractions recorded in both the orad and aborad chambers. In separate experiments, the effects of addition to the orad chamber of high (10-4 M) and low (10-8 M) concentrations of nifedipine, 0 Ca2+, 20 mM Mg2+ Krebs solution, and atropine (10-6 M) on both BD and EFS responses in the orad and aborad chambers were determined.

The Krebs solution in the aborad chamber was changed every 20-30 min throughout the duration of the protocol. Responses at all recording sites were analyzed in both chambers; however, for clarity, data presented from the aborad chamber are from the 3-cm site, as the response at this site was representative of the two other aborad sites.

Esophageal Compliance: Barostat Studies

To further investigate the mechanism of action of these inhibitors of smooth muscle contractility and neurotransmission, esophageal compliance was assessed using an automated barostat (SVS Barostat, Synectics visceral stimulator module) and data acquisition system (Polygram for Windows version 1.0B6).

A compliant 8-cm-long polyethylene bag (maximum diameter, 30 mm; maximum capacity, 50 ml) was employed to measure in vitro changes in esophageal compliance in response to agents that affect smooth muscle contractility and neurotransmission. Before each experiment, the intraesophageal bag was tested for leaks and calibrated manually. The compliance of the bag ex vivo was measured to ensure reliability of our measurements in the low-compliance portion of the curve. This confirmed that, with operating volumes of <20 ml, the elastic recoil of the bag did not contribute to the resistance to inflation. This allowed us to be certain that any change in volume for a given pressure change primarily reflected the esophageal wall properties and not those of the distending polyethylene bag.

After an 8- to 10-cm segment of distal esophagus was removed from the opossum, it was placed in a tissue bath, and the barostat bag was inserted into the esophagus and secured in place with a tie at both the proximal and distal ends. The tissue was then left for a 20-min stabilization period. The selected agent (10-4 M nifedipine, 0 Ca 2+-20 mM Mg 2+ Krebs, or 10-6 M TTX ) or control (normal Krebs solution) was then added to the bath and the protocol commenced. The intraesophageal bag was inflated to the selected distending pressure for 30 s, followed by a 60-s deflation period. The distending pressures employed were in the following order: 0, 1, 2, 3, 4, 8, 12, 16, and 20 mmHg. This distension cycle took 12 min and was followed by a 3-min rest period. The protocol was repeated four times each hour for 2 h. During bag distension, the pressure in the bag is maintained at a constant level while changes in barostat bag volume are monitored. Hence, at a selected distending pressure, any change in intraesophageal bag volume is indicative of a change in the compliance of the esophageal wall. Thus an increase in bag volume indicates an increase in compliance, whereas a decrease in bag volume indicates a decrease in compliance.

For each experimental condition, a volume-pressure curve was established and analyzed using regression analysis. As described by Mayrand and Diamant (12), the two aspects of tone that were assessed were 1) compliance of the esophageal wall, expressed as the slope of the volume-pressure relationship (dV/dP), and 2) resistance of the esophageal wall to initial stretch, expressed as the x-intercept of the volume-pressure graph. The equation of a linear function can be represented as y = A + Bx, where B is the slope and A is the y-intercept. The x-axis intercept (when y = 0) is defined by the ratio -A/B. The maximum volume recorded at the selected distending pressure was used to plot the pressure-volume curves in all of the experiments.

Phasic contractions over the top of the barostat bag were also identified as rapid increases in the preset intraesophageal bag pressure. The barostat is equipped with feedback sensors that act to offset pressure variations by adjusting the volume of air injected into the intraesophageal bag. These real-time barostat volume adjustments serve to maintain constant distending pressure on the esophageal wall. A contraction index (CI) was defined to quantify the phasic contractile activity over the top of the barostat bag during one complete distension cycle. The amplitude of the contraction as measured by volume displacement of the barostat bag (ml) × the duration of the contraction (s) is a measure of phasic contractility. The CI is the cumulative sum of the contractile activity for all the selected distending pressures in one distension cycle.

Drugs

Nifedipine, TTX, and atropine were supplied by Sigma Chemical.

Statistical Methods

In the double-chambered organ bath studies, comparison of the magnitude of contraction amplitudes before and after addition of selected agents was carried out using a two-tailed paired Student's t-test. For the barostat studies, the equations defining the linear relationship between volume and pressure were calculated by linear regression analysis using the least-squares principle (Microsoft Excel for Windows 95 version 7). Differences between treatment groups were determined by one-way ANOVA. When significant variation was detected, the Student-Newman-Keuls post test for multiple-sample comparison was carried out. P < 0.05 was accepted as indicative of a statistically significant difference between two sets of observations. All values reported are means ± SE. All statistical calculations were done using the GraphPad Instat version 2.04, a statistical package (GraphPad Software).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Descending Peristaltic Reflex Studies

A typical response to intramural BD and EFS in the opossum smooth muscle esophagus in vitro is depicted in Fig. 2. Off contractions are seen at sites distal to the balloon after balloon deflation or EFS. This reflex was usually reproducible for several hours, although the magnitude of the response tended to gradually decrease over time. The addition of TTX (10-6 M) to the orad chamber abolished both the BD- and EFS-induced descending peristaltic reflex (n = 5).


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Fig. 2.   Typical response to BD (A) and EFS (B) of the opossum smooth muscle esophagus in vitro. Circular muscle "off" contractions are seen at sites distal to the balloon on deflation and after EFS. Addition of nifedipine to the orad chamber resulted in inhibition of the off contractions in both the orad and aborad chambers in response to BD (C). With EFS (D), only responses in the orad chamber were inhibited.

Effect of nifedipine. When added to the orad chamber in cumulative concentrations ranging from 10-8 to 10-4 M, nifedipine inhibited phasic off contractions in both the orad and aborad chambers in a concentration-dependent manner (Fig. 3). When added to the orad chamber in supramaximal concentrations (10-4 M; n = 5), nifedipine virtually abolished the BD-induced off contractions in both the orad (55.1 ± 7.3 vs. 0.4 ± 0.2 mmHg; P = 0.0016) (Fig. 4A) and aborad chambers (42.8 ± 8.9 vs. 1.7 ± 0.9 mmHg; P = 0.0096) (Fig. 4B). Furthermore, larger BD volumes (i.e., 3 ml) were unable to initiate this inhibited reflex response after nifedipine. However, with EFS (n = 5), supramaximal concentrations of nifedipine markedly inhibited the off contractions in the orad chamber (50.9 ± 5.1 vs. 9.0 ± 3.3 mmHg; P = 0.005) (Fig. 5A), but unlike BD had no effect on contractions in the aborad chamber (41.4 ± 7.3 vs. 33.4 ± 18.5 mmHg; P = 0.52) (Fig. 5B). Low concentrations of nifedipine (10-8 M) added to the orad chamber significantly decreased the amplitude of BD-induced off contractions in the orad chamber (35.3 ± 4.4 vs. 17.3 ± 9.7 mmHg; P = 0.024) (Fig. 4A), but did not significantly decrease contraction amplitude in the aborad chamber (34.3 ± 5.9 vs. 15.3 ± 5.6 mmHg; P = 0.078). Low concentrations of nifedipine (10-8 M) in the orad chamber also did not significantly affect the EFS-induced off contractions in either chamber (Fig. 5). The inhibitory effect of nifedipine was irreversible as the off response did not return after several washes of the tissue with fresh Krebs solution.


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Fig. 3.   Concentration-response curves depicting the effect of nifedipine (added to orad chamber) on BD-induced off contractions in both the orad (black-triangle) and aborad chambers ().



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Fig. 4.   Effect of selected agents, added to the orad chamber, on the BD-induced descending peristaltic reflex. A: addition to the orad chamber of either Ca2+-free Krebs or nifedipine (10-8 and 10-4 M) significantly inhibited off contractions in the orad chamber. B: addition of either Ca2+-free Krebs or nifedipine (10-4 M) to the orad chamber also significantly inhibited off contractions in the aborad chamber. Nifedipine (10-8 M), added to the orad chamber, decreased amplitude of off contractions in the aborad chamber, but this did not reach statistical significance (P = 0.078). Addition of atropine (10-6 M) to the orad chamber did not affect off contractions in either the orad or aborad chambers. Drug-treated and control responses are represented by open and filled bars, respectively; n = 5-7 for each drug studied. * P < 0.025, ** P < 0.01 vs. control observations by 2-tailed paired Student's t-test.



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Fig. 5.   Effect of selected agents, added to the orad chamber, on the EFS-induced descending peristaltic reflex. A: both Ca2+-free Krebs and nifedipine (10-4 M) significantly inhibited the neurally mediated off response in the orad chamber. The response after atropine (10-4 M) or nifedipine (10-8 M) was not statistically different from control values. B: when added to the orad chamber, Ca2+-free Krebs, nifedipine (10-8 or 10-4 M), or atropine (10-6 M) did not significantly inhibit the contractions in the aborad chamber. Drug-treated and control responses are represented by open and filled bars, respectively; n = 5-7 for each drug studied. * P < 0.05, ** P < 0.01 vs. control observations by 2-tailed paired Student's t-test.

Effect of 0 Ca2+-20 mM Mg2+ Krebs. Substitution of normal Krebs solution with 0 Ca 2+-20 mM Mg 2+ Krebs solution (n = 5) to the orad chamber markedly inhibited the BD-induced off contractions in the orad chamber (61.2 ± 15.1 vs. 2.0 ± 1.2 mmHg; P = 0.018) (Fig. 4A) and aborad chamber (55.0 ± 7.1 vs. 10.8 ± 6.8 mmHg; P = 0.014) (Fig. 4B). Moreover, larger BD volumes of 3 ml were unable to initiate this inhibited reflex response. Although EFS-induced contractions (n = 5) were significantly attenuated in the orad chamber (39.6 ± 9.5 vs. 8.5 ± 3.0 mmHg; P = 0.049) (Fig. 5A), the contractions were not significantly inhibited in the aborad chamber (50.0 ± 7.2 vs. 32.8 ± 18.7 mmHg; P = 0.44) (Fig. 5B). The inhibitory effect of 0 Ca2+-20 mM Mg2+ Krebs solution was reversible, with postwash contraction amplitudes returning to control levels (n = 5).

Effect of atropine. Addition of atropine (10-6 M; n = 5) to the orad chamber did not significantly affect the BD- or EFS-induced off contractions in either chamber.

Barostat Studies

Effect of smooth muscle relaxants and neural blockade on esophageal compliance. Linear regression analysis of pressure-volume curves of the smooth muscle esophagus demonstrated a high degree of linearity in all experimental treatment groups (Fig. 6). Furthermore, there were no significant differences (P = 0.91) between the slopes of the pressure-volume curves in any of these treatment groups.


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Fig. 6.   Effect of selected agents on esophageal body pressure-volume curves. The slope of the pressure-volume curves as established with the electronic barostat was no different between any of the treatment groups compared with controls: controls (A); Ca2+-free Krebs (B); nifedipine (C); TTX (D) (P = 0.91, ANOVA).

Resistance of esophageal wall to initial stretch. The values of the x-axis intercept, which represent the resistance of the esophageal wall to initial stretch, were extrapolated from the pressure-volume curves of the treatment groups and are shown in Fig. 7. As determined by ANOVA, there were no significant differences (P = 0.71) among the x-axis intercepts between any of treatment groups.


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Fig. 7.   Effect of selected agents on resistance of esophageal wall to initial stretch. The resistance to initial stretch, as extrapolated from the x-intercept from the pressure-volume curves, is a reflection of tone in the resting state. This point represents the level at which the distending pressure inside the barostat bag is equal to the pressure outside the bag and corresponds to the minimum amount of pressure required to distend (or open) the esophagus. There were no significant differences in x-intercept values or resistance to initial stretch among any of the treatment groups compared with controls: controls (A); Ca2+-free Krebs (B); nifedipine (C); TTX (D) (P = 0.71, ANOVA).

Phasic contractions over top of distending bag. With inflation of the barostat bag to pressures of at least 3 mmHg, phasic contractile activity was induced in the esophagus. To quantify the inhibition of phasic contractility in response to these agents, both the contraction frequency and CI were examined. A minimum distending pressure of 3 mmHg was required to initiate phasic contractions over the top of the barostat bag. Both contraction frequency and CI peaked at a barostat bag distending pressure of 8 mmHg and then decreased as the distending pressure increased to 20 mmHg. As depicted in Fig. 8, with respect to the control group (132 ± 24 ml · s), the CI was significantly inhibited by nifedipine (1 ± 0 ml · s, P < 0.001) and 0 Ca2+ -20 mM Mg2+ Krebs solution (1 ± 0 ml · s, P < 0.001). However, neural blockade with TTX (91 ± 14 ml · s) did not significantly alter the overall CI. The frequency of contractions during a complete distension cycle was also investigated. With respect to the control group (12.8 ± 2.0 contractions per distension cycle), the contraction frequency was significantly inhibited by nifedipine (0.3 ± 0.0, P < 0.01) and 0 Ca2+-20 mM Mg2+ Krebs solution (0.0 ± 0.0, P < 0.01). However, neural blockade with TTX did not significantly alter the overall contraction frequency (9.2 ± 2.9, P > 0.05). Further quantitative analysis revealed that there was a significant increase in initial contraction amplitude over the top of the barostat bag between control and TTX experiments, as demonstrated by barostat bag displacement volume at distending bag pressures of both 8 (4.6 ± 0.7 vs. 6.6 ± 0.7 ml; n = 5; P = 0.01) and 12 mmHg (4.2 ± 0.4 vs. 6.8 ± 0.9 ml; n = 5; P = 0.007). However, at these distending pressures, there were no differences between control and TTX experiments in either the duration of the initial contraction (8 mmHg: 3.8 ± 0.5 vs. 3.6 ± 0.4 s, P = 0.77; 12 mmHg: 3.8 ± 0.4 vs. 3.3 ± 0.3 s, P = 0.35) or latency to onset of the initial contraction (8 mmHg: 1.2 ± 0.1 vs. 2.7 ± 0.7 s, P = 0.10; 12 mmHg: 1.5 ± 0.3 vs. 2.5 ± 0.4 s, P = 0.08).


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Fig. 8.   Effect of selected agents on contraction index. The contraction index [defined as amplitude of contraction (ml) × duration (s) per distension cycle] was significantly inhibited by Ca2+-free Krebs (n = 4; B) and nifedipine (10-4 M, n = 4; C). However, neural blockade with TTX (10-6 M, n = 5; D) did not significantly inhibit the contraction index with respect to control values (A), indicating that these contractions are largely myogenic. ** P < 0.001 vs. control observations; n = 5.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The distension-induced descending peristaltic reflex is a highly complex motor activity that is influenced by central and peripheral levels of control as well as myogenic properties of the musculature. Though this reflex has been extensively studied in the small intestine, little is known about the intramural mechanisms responsible for the initiation and propagation of this reflex in the smooth muscle esophagus. As previously documented (16) in triple-chamber organ bath studies, addition of 0 Ca2+-20 mM Mg2+ Krebs to the orad chamber (i.e., site of distension) abolished the distension-induced descending peristaltic reflex. However, it was not clear from these earlier studies (16) whether this inhibition was due to synaptic blockade or impairment of muscle contractility. The current experiments support the latter.

In the present study, the dihydropyridine nifedipine was used to selectively impair influx of extracellular Ca2+ into the smooth muscle cells and hence inhibit esophageal smooth muscle contractility without affecting synaptic transmission. Although no studies could be found that investigated the effects of nifedipine on synaptic transmission in the opossum, studies in the guinea pig ileum have demonstrated that blockade of L-type Ca2+ channels with either verapamil or nifedipine does not impede synaptic transmission (11). Ca2+ channel blockers, including nifedipine, are widely used to treat spastic disorders of the esophagus, including nutcracker esophagus and diffuse esophageal spasm. In humans, they have been shown (2, 18) to reduce lower esophageal sphincter (LES) tone and the amplitude and duration of peristaltic esophageal contractions. In our preparation, addition of nifedipine to the orad chamber virtually abolished the balloon deflation-related off contractions in both the orad and aborad chambers, but even in supramaximal concentrations it inhibited the EFS-related off contractions in the orad chamber only. This inhibitory effect of nifedipine was not reversible, as responses did not return after several washes with Krebs solution, even after 1 h. Although we did not attempt to reverse the effect of nifedipine by increasing the Ca2+ content in our preparation, previous in vitro studies (8) in the small intestine have demonstrated that the inhibitory effect of nifedipine on intestinal contractility is not reversed by increasing Ca2+ concentration in the organ bath. It is unlikely that nifedipine affected neurotransmission in the descending reflex pathway, because EFS of intramural nerves in the orad chamber was still able to initiate the reflex as demonstrated by off contractions in the aborad chamber. These results indicate that local smooth muscle contraction at the site of distension is an important component of the distension-induced descending peristaltic reflex in the esophagus.

To test whether blockade of cholinergic neuromuscular transmission would induce effects similar to nifedipine on the phasic off contraction, atropine was placed in the orad stimulating chamber. This had no effect on the BD- or EFS-induced off contractions in either the orad or aborad chambers. The lack of effect of atropine on the orad chamber response is in keeping with previous studies (4, 16) demonstrating that off contractions of esophageal body circular muscle are noncholinergic. The fact that atropine in the orad chamber also did not affect off contractions in the aborad chamber excludes a role for either muscarinic neurotransmission or cholinergically mediated "on" contractions in the distension-induced descending peristaltic reflex.

The objective of the barostat studies in the smooth muscle esophagus was to assess the mechanism by which the selected agents were inhibiting the descending peristaltic reflex. We initially hypothesized that tonic muscle contraction occurring in response to esophageal distension was critical to the initiation of the descending peristaltic reflex and that this tone was inhibited by nifedipine and 0 Ca2+-20 mM Mg2+ Krebs solution. Interestingly, we could not detect any significant differences in esophageal compliance or resistance of the esophageal wall to initial stretch in any of the treatment groups. This finding suggests that these agents do not alter resting tone in the esophageal body and that the effect of these agents on the distension-induced descending peristaltic reflex was not due to inhibition of tonic esophageal contraction in response to stretch. These results are consistent with other barostat studies (18) demonstrating that nifedipine does not alter resting esophageal body tone in vivo in humans. This is in contrast to the LES, in which nifedipine inhibits tonic contractility in a dose-dependent manner (2).

These results suggest that there may be no active tonic contraction of the esophageal body of the opossum in vitro either at rest or in response to distension. This is consistent with studies (14) in humans where no measurable tone was found in the esophageal body at rest. Furthermore, Tøttrup and colleagues (19) have demonstrated that in vitro circular smooth muscle strip preparations from the cat esophageal body show little resting tone under isometric conditions. However, in vivo barostat studies in the human have demonstrated that smooth muscle tone (both compliance and resistance to initial stretch) has an active component that can be reduced by the smooth muscle relaxant amyl nitrate (6, 13). We could not confirm active tone in the opossum esophagus using other pharmacological methods of relaxation.

One explanation for our results is that the resistance to stretch in the esophagus is due primarily to passive viscoelastic properties of the esophageal wall. For instance, there are localized differences in composition of smooth muscle in the esophagus, such as varied levels of collagen and elastic fibers, and these may be contributing factors to the compliance of the esophagus (9, 10).

Although tonic muscle activity was not altered in these experiments, phasic contractions over the top of the distending barostat bag were significantly inhibited by nifedipine and 0 Ca2+-20 mM Mg2+ Krebs solution. However, TTX did not significantly inhibit these phasic contractions, indicating that they are largely myogenic in origin. Previous BD studies (15) in the opossum esophagus in vivo recorded smooth muscle contractions over the top of the distending balloon during the period of BD. The amplitude of these contractions increased with exposure to TTX. This not only suggests that these contractions are myogenic in origin, but also that, under normal circumstances, the muscle receives some tonic inhibitory input that is removed when TTX is applied. Christensen (4) also observed similar TTX-resistant contractions at and/or just proximal to a distending balloon. It is important to recognize that unlike the intraesophageal balloons used by Christensen (4) and Paterson (15), which were 2 cm in length and inflated for ~5 s, the phasic contractions recorded in this current protocol were from an 8-cm-long bag that was inflated for 30 s. Hence the contractile activity observed in these experiments represents a composite (i.e., both myogenic and neurogenic) of esophageal contractions in response to prolonged bag distension over a long (8-cm) segment. Although the types of phasic contractions were not assessed, it is interesting to note that the amplitude of the initial contraction observed in response to barostat distension was significantly enhanced in the presence of TTX, suggesting that this contractile response is functionally similar to the previously described myogenic contractions observed over the top of the distending balloon (15).

A recent report by Brookes and colleagues (3) examined the role of smooth muscle excitability in the initiation of peristalsis in the guinea pig ileum. In these in vitro experiments (3), segments of intestine were radially stretched using a microprocessor-controlled stepper motor with an in-series force transducer to record circular muscle contraction. With addition to this preparation of agents that reduce the excitability of smooth muscle, an increase in the threshold stretch required to initiate peristalsis was observed. Addition of agents that selectively increase the excitability of circular muscle resulted in a reduction in threshold stretch required for peristaltic initiation. Furthermore, these experiments demonstrated that, when the ileum is close to threshold stretch, peristalsis can be triggered simply by increasing circular muscle tension. These results suggest that the initiation of peristalsis may be due to initial contraction of circular muscle activating neuronal circuitry via contraction-sensitive mechanoreceptors.

In summary, our experiments demonstrate that the descending peristaltic reflex is markedly inhibited when agents affecting smooth muscle contractility, but not synaptic transmission, are placed at the site of distension. These agents do not alter the compliance of the smooth muscle esophageal body or its resistance to initial stretch, but inhibit phasic contraction over the top of the distending barostat bag. Taken together, these observations suggest that local stretch-induced phasic muscle contractions are involved in the initiation of the BD-induced descending peristaltic reflex in the opossum smooth muscle esophagus. When combined with the previous results of Paterson and Indrakrishnan (16), these observations support the hypothesis that the distension-induced descending peristaltic reflex involves long descending intramural neurons that may have both mechanosensory and motor functions. That is, local stretch-induced phasic muscle contraction may directly activate long descending intramural neurons, which then release NO onto circular smooth muscle downstream. This in turn produces hyperpolarization (inhibition) of the muscle followed by "rebound" contraction (excitation). Confirmation of this hypothesis, however, must await anatomic demonstration of such neurons.


    ACKNOWLEDGEMENTS

We thank David Miller for technical assistance and Brenda De Longhi for typing the manuscript.


    FOOTNOTES

This work was supported by Medical Research Council of Canada Grant MA-9978.

Address for reprint requests and other correspondence: W.G. Paterson, Gastrointestinal Diseases Research Unit and Depts. of Medicine and Physiology, Queen's Univ., Kingston, Ontario, Canada K7L 5G2 (E-mail: patersow{at}hdh.kari.net).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 21 October 1999; accepted in final form 25 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Gastrointest Liver Physiol 280(3):G431-G438
0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society




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