Biphasic relaxation of the opossum lower esophageal sphincter: roles of NO ·, VIP, and CGRP

Aliye Uc1, S. T. Oh2, Joseph A. Murray3, Eugene Clark3, and Jeffrey L. Conklin3

Departments of 3 Internal Medicine, 1 Pediatrics, and 2 Surgery, University of Iowa College of Medicine and Department of Veterans Affairs Medical Center, Iowa City, Iowa 52242


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Vasoactive intestinal polypeptide (VIP) and nitric oxide (NO ·) are thought to mediate lower esophageal sphincter (LES) relaxation. Transverse muscle strips from the opossum LES were used to test this hypothesis. Electrical field stimulation (EFS) produced a biphasic LES relaxation: a rapid component during the stimulus was more prominent at lower stimulus frequencies, and a sustained component was more prominent at higher frequencies. Nomega -nitro-L-arginine and hemoglobin inhibited the rapid component but affected the sustained component less. Exogenous VIP decreased LES tone. A number of purported VIP antagonists blocked neither VIP-induced nor EFS-induced relaxation of the LES. The calcitonin gene-related peptide (CGRP) antagonist CGRP-(8---37) did not alter EFS-induced LES relaxation. EFS-induced relaxation of opossum LES muscle is biphasic, and the initial, rapid component of the relaxation is mediated primarily by NO ·. The mediator of the sustained component was not identified.

nitric oxide; gastrointestinal motility; smooth muscle; enteric nervous system; vasoactive intestinal polypeptide; calcitonin gene-related peptide


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CIRCULAR SMOOTH MUSCLE FROM the esophageal body generates little if any tone at rest, whereas circular smooth muscle from the lower esophageal sphincter (LES) is tonically contracted. In response to swallowing, a peristaltic contraction sweeps down the length of the esophagus and the LES relaxes. Nitric oxide (NO ·) (25, 33, 34, 37) and vasoactive intestinal polypeptide (VIP) (3, 4, 15, 30) are proposed as neurotransmitters that control LES relaxation and/or esophageal peristalsis. Each causes LES relaxation, both VIP and NO · can be released from esophageal nerves with an appropriate stimulus, and nitric oxide synthase (NOS) and VIP are found in myenteric neurons that innervate the circular smooth muscle of the esophagus.

The relative roles that VIP and NO play in the nerve-induced relaxation of the LES remain controversial. An older literature supports the hypothesis that VIP is the mediator of nerve-induced relaxation of the LES (4, 15); anti-VIP antibodies inhibited LES relaxation produced either by swallowing or by stimulating intrinsic esophageal nerves in vitro. More recent studies indicate that NO ·, not VIP, is the major mediator of nerve-induced LES relaxation (10, 12, 25, 26, 34). In these earlier studies, Nomega -nitro-L-arginine (L-NNA), an inhibitor of NOS, and hemoglobin, an NO · scavenger, both inhibited nerve-induced LES relaxation. In addition, hemoglobin markedly attenuated the LES relaxation that follows swallowing (10, 26). One group of investigators has taken both approaches and suggests that the response of LES muscle to nerve stimulation depends on the integrated output of three innervations: an NO innervation, a cholinergic innervation, and a VIP innervation (27).

The aims of this study were to test the hypothesis that VIP and NO interact to control the nerve-mediated relaxation of the LES and hopefully to clear up some of the confusion regarding the roles of these agents in the control of esophageal motor function.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Fasted adult opossums of either sex weighing 2-4 kg were anesthetized with ketamine HCl (30 mg/kg)-acepromazine (0.3 mg/kg) given intramuscularly and pentobarbital sodium (50 mg/kg) given intraperitoneally. The chest and abdomen were opened, and the entire intrathoracic and intra-abdominal esophagus was marked and measured in situ. The esophagus was transected at the proximal mark and excised along with a cuff of gastric tissue. The entire tissue was opened in the long axis of the esophagus along the lesser curve of the stomach. It was washed with warmed, aerated Krebs solution and pinned flat in a tissue bath at its dimensions in situ with the mucosal surface facing up. The bath contained oxygenated (95% O2-5% CO2) Krebs solution maintained at 37°C and pH 7.4. The mucosa and most of the submucosa were removed, and the LES was recognized as a thickened band of circular muscle at the gastroesophageal junction. Transversely oriented strips measuring 2 cm × 0.2-0.3 cm were prepared from the LES so that the long axis of the muscle strip paralleled the long axis of smooth muscle cells constituting the circular muscle layer. Muscle strips were attached with silk suture to force displacement transducers, positioned between platinum electrodes placed 4 mm apart, and lowered into 8-ml jacketed tissue baths filled with Krebs solution maintained at 37°C and bubbled continuously with 95% O2-5% CO2. The electrodes were connected to the output of a Grass S11 stimulator that delivered 4-s trains of 0.5 ms, 50-V square wave pulses at 1-20 Hz. These stimulus parameters were previously shown to produce activation of intrinsic esophageal nerves (9, 25). Additional studies were done with continuous stimulation of the nerves for 2 min at 10 Hz as described in a recent article (27). Each force-displacement transducer was attached to a rack-and-pinion device that allowed sequential stretching of the muscle strips. The output of the force-displacement transducers was processed through MacLab 8 analog-to-digital converter and recorded on Macintosh IICi computer.

Each muscle strip was stretched rapidly until 100 mg of force was generated. This was taken as the initial length. Muscle strips were then sequentially stretched to 130% of initial length. The strips were equilibrated for 1 h in the warmed, oxygenated Krebs solution before experimentation. Only LES strips generated tone at rest and relaxed on stimulation. Muscle strips were stimulated at 5-min intervals beginning 15 min before experimentation. Only muscle strips showing reproducible responses to electrical field stimulation (EFS) were used. All drug concentrations are final concentrations in the tissue bath. The tissue baths were coated with Sigmacote because of excessive foaming with BSA.

Solutions and drugs. The modified Krebs solution used in these experiments contained (in mM) 138.5 Na+, 4.6 K+, 2.5 Ca2+, 1.2 Mg+, 125 Cl-, 21.9 HCO-3, 1.2 H2PO-4, 1.2 SO-4, and 11.5 glucose. This solution was maintained at 37°C and bubbled continuously with 95% O2-5% CO2 to maintain a pH of 7.4 throughout the experiment. Ketamine was obtained from Aveco (Fort Dodge, IA). Pentobarbital sodium was obtained from the University of Iowa Pharmaceutical Service. The following agents were purchased from Sigma Chemical (St. Louis, MO): aprotonin, bacitracin, VIP, L-NNA, L-arginine, TTX, [Lys1,Pro2,5,Arg3,4,Tyr6]VIP (Sigma V2131) and VIP-(10---28) (Sigma V5381), VIP-(6---28) (Sigma V4508), [D-p-Cl-Phe6,Leu17]VIP (Sigma V4380), atropine sulfate, carbachol, BSA, and Sigmacote. [Ac-Tyr1,D-Phe2]growth hormone-releasing factor-(1---29) amide (Peninsula 8076) was purchased from Peninsula; VIP antiserum (64-720-1) and rabbit serum (64-291) were purchased from ICN Biochemicals (Aurora, OH).

One milliliter of VIP antiserum or rabbit serum was dialyzed against 3 × 4,000 ml of 1× PBS at 4°C by using Spectra/por molecular porous membrane (132655) (molecular weight cutoff of 6,000-8,000).

Data analysis. All physiological recordings were made and analyzed with MacLab software. Data are expressed as means ± SE; n represents the number of animals from which observations were made. Student's t-test or two-way ANOVA was used when appropriate to determine levels of statistical significance. The Finney method was used for the calculation of EC50 values.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Response of the LES to varying frequencies of electrical stimulation. Muscle strips from the LES maintained tone at rest and relaxed when stimulated by an electrical field: 4-s trains of square wave pulses with a duration of 0.5 ms, an amplitude of 50 V, and a rate of 1-20 Hz (Fig. 1). This relaxation appeared to have two components, the presence of which depended on the frequency of the stimulus (Fig. 1). At lower frequencies of stimulation, an early, rapid phasic component of the relaxation was apparent during the stimulus. As the frequency of the stimulus was increased, a second more sustained relaxation that persisted after the stimulus became apparent. The appearance of these responses varied somewhat from experiment to experiment; on some occasions, there was a brief contraction interposed between the fast and slow components of the relaxation.


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Fig. 1.   Effect of Nomega -nitro-L-arginine (L-NNA) on the biphasic relaxation of the lower esophageal sphincter (LES). A, left: control, demonstrating a recording of the frequency-response relationship for electrical field stimulation (EFS)-induced relaxation of LES. Tension generated by the muscle (g) is shown, and horizontal bar indicates time. Stimulus artifacts indicating the timing of EFS with 4-s trains of 0.5 ms, 50-V square wave pulses at 1-20 Hz are shown on x-axis. Note that there are 2 components to the LES relaxation, a rapid initial component that occurs during EFS and a more prolonged second component that is sustained after the EFS. The second component appears to become more prominent as the stimulus frequency increases. B, left: same frequency-response relationship after tissue was exposed to 0.3 mM L-NNA for 30 min. Note the apparent loss of the rapid component of the relaxation. B, right: response of this tissue to a 20-Hz stimulus 10 min after it was exposed to 1 µM TTX. Response appears to result from activation of the intrinsic innervation of the LES. A, right: overlay of the response to a 20 Hz stimulus in the absence and presence of 0.3 mM L-NNA.

Effect of L-NNA and hemoglobin on the EFS-induced LES relaxation. In a number of previous studies, we and others demonstrated that antagonists of NOS are able to inhibit EFS-induced relaxation of the LES (13, 25, 27, 33, 34, 37). Because most previous studies of EFS-induced relaxation of the LES focused on the early, more rapid component of the relaxation, little is known about the neuromuscular mechanisms producing the slower component of the relaxation. L-NNA, a competitive antagonist of NOS, was used to test the hypothesis that both components of EFS-induced relaxation are dependent on NO ·. The rapid component of the EFS-induced relaxation appeared to be completely antagonized by 0.3 mM L-NNA, but the slower component seemed to be much less affected by inhibition of NOS (Figs. 1 and 2). In fact, at the highest stimulus frequency used in these studies, L-NNA appeared to have almost no effect on the slower component of the relaxation (Figs. 1 and 2).


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Fig. 2.   Effect of L-NNA on the biphasic relaxation of the LES. A: graphic demonstration of the frequency-response relationship for the initial, rapid LES relaxation that occurs during EFS. , Control responses. , Responses after tissue was exposed to 0.3 mM L-NNA for 30 min. Error bars indicate SE (n = 11). * P < 0.05. Note that the relaxation during EFS is essentially abolished by L-NNA. B: frequency-response relationship for the second, more sustained relaxation that persists after EFS. , Control responses. , Responses after tissue was exposed to 0.3 mM L-NNA for 30 min. Error bars indicate SE (n = 11). * P < 0.05. Note that L-NNA has progressively less effect on this response as the frequency of stimulation increases, until at 20 Hz it appears to have no effect.

TTX (1 µM) antagonized both the rapid and the slow components of the relaxation. At the maximum stimulus rate of 20 Hz, the rapid component of the relaxation diminished from 65.4 ± 7.5% to 0.0 ± 0.0% (means ± SE, P < 0.01, n = 4). After the slower component of the relaxation was isolated with L-NNA, the residual relaxation decreased from 29.9 ± 10.2% to 3.2 ± 1.9% (means ± SE, P < 0.05, n = 4) (Fig. 1).

Hemoglobin, which we know antagonizes EFS-induced relaxation by scavenging NO · (10), was also used to determine the role played by NO · in the genesis of both components of the LES relaxation. The rapid component of the EFS-induced relaxation was decreased by >50% by 0.1 mM hemoglobin, but it had less effect on the slower component of the relaxation, especially at higher stimulus frequencies, which produce a more prominent slow component of the relaxation (Fig. 3).


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Fig. 3.   Effect of hemoglobin (Hgb) on the biphasic relaxation of the LES. A: relaxation of the LES caused by EFS (4-s trains of 0.5 ms, 50-V square wave pulses at 20 Hz) in the absence and presence of 0.1 mM hemoglobin, also showing composite of these 2 responses. Horizontal bars, which also represent 4 s, indicate the timing of the EFS. B: graphic demonstration of the frequency-response relationship for the initial, rapid LES relaxation that occurs during EFS. , Control responses. , Responses after tissue was exposed to 0.1 mM hemoglobin for 30 min. Error bars indicate SE (n = 5). Note that hemoglobin attenuates the relaxation during EFS. C: frequency-response relationship for the second, more sustained relaxation that persists after EFS. , Control responses. , Responses after tissue was exposed to 0.1 mM hemoglobin for 30 min. Error bars indicate SE (n = 5). * P < 0.05. Note that hemoglobin has progressively less effect on this response as the frequency of stimulation increases, until at 20 Hz it appears to have no effect.

Effect of L-NNA and hemoglobin on VIP-induced LES relaxation. VIP relaxed circular muscle strips from the LES in a concentration-dependent manner (Fig. 4). To test the hypothesis that VIP may be causing the release of an NO-containing transmitter substance, we explored the effect of L-NNA and hemoglobin on the VIP concentration-response relationship. Incubation of the muscle with 0.5 mM L-NNA had no significant effect on VIP-induced relaxation (VIP EC50 = 56 ± 16 nM vs. EC50 = 42 ± 17 nM for VIP plus 0.5 mM L-NNA; P > 0.05, n = 11) (Fig. 4).


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Fig. 4.   Effect of L-NNA or hemoglobin on the LES relaxation produced by varying concentrations of vasoactive intestinal polypeptide (VIP). A: VIP concentration-response relationship in response to VIP alone () and in the presence of 0.5 mM L-NNA (). B: VIP concentration-response to VIP alone () and in the presence of 0.1 mM hemoglobin (), a concentration of hemoglobin that produces a >50% inhibition of nerve-induced and nitric oxide-induced LES relaxation. Note that neither L-NNA nor hemoglobin inhibited the VIP-induced LES relaxation.

In previous experiments, we demonstrated that 0.1 mM hemoglobin produces more than a 50% inhibition of both NO ·-induced and nerve-induced relaxation of the LES (10). Hemoglobin did not alter the VIP concentration-response relationship (VIP EC50 = 36 ± 3 nM vs. EC50 = 33 ± 5 nM for VIP plus 0.1 mM hemoglobin; P > 0.05, n = 6) (Fig. 4).

As a test of the hypothesis that VIP causes LES relaxation by stimulating the release of another inhibitory neurotransmitter, we observed the effect of VIP on LES tissues pretreated with the neurotoxin TTX. TTX (1 µM) produced near-complete blockade of nerve-induced relaxation of the LES but did not attenuate relaxation in response to exogenous 0.1 µM VIP. In fact, the percent relaxation induced by 0.1 µM VIP increased slightly in the presence of TTX, but the increase was not statistically significant. TTX increased LES tone in some experiments, but this was not a consistent finding.

Effects of the VIP antagonists on VIP-induced and nerve-induced LES relaxation. We attempted to use the known VIP antagonists [Lys1,Pro2,5,Arg3,4,Tyr6]VIP (Sigma V2131), VIP-(10---28) (Sigma V5381), VIP-(6---28) (Sigma V4508), [D-p-Cl-Phe6,Leu17]VIP (Sigma V4380), and [Ac-Tyr1,D-Phe2]growth hormone-releasing factor-(1---29) amide (Peninsula 8076) to test the hypothesis that VIP released from intrinsic esophageal nerves during EFS mediates LES relaxation. All of these agents produced a transient increase in LES tone that was not attenuated by 1 µM atropine. Of these agents, [Lys1,Pro2,5,Arg3,4,Tyr6]VIP and VIP-(10---28) proved to be inconsistent inhibitors of VIP-induced LES relaxation. The other purported VIP antagonists did not antagonize the VIP-induced relaxation. These experiments were carried out both with and without protease inhibitors (0.1% BSA, 4 µg/ml aprotonin, and 20 µM bacitracin) in the tissue bath to determine if the inconsistency of the results was due to rapid breakdown of either the VIP or the VIP antagonists. The presence of these protease inhibitors did not affect the results. Those antagonists that inconsistently antagonized the effects of VIP, [Lys1,Pro2,5,Arg3,4,Tyr6]VIP and VIP-(10---28), had no discernible effect on the amplitude of either the rapid or slow component of nerve-induced LES relaxation.

Effects of VIP antiserum and rabbit serum on LES resting tone and nerve-induced relaxation. In a recent study, Ny et al. (27) explored the effect of a VIP antiserum on the neuromuscular function of circular smooth muscle from the LES of the cat. In their studies, VIP antiserum at a 1:25 dilution abolished the tone in spontaneously contracted LES muscle. LES muscle that did not generate spontaneous tone was precontracted with 1 µM carbachol and then exposed to VIP antiserum at a 1:25 dilution. Although the authors did not comment on the effect of the antiserum on carbachol-induced tone, they noted that it produced a 60-100% inhibition of nerve-induced LES relaxation and abolished the relaxation produced by VIP.

Using the same VIP antiserum, purchased from another vender [VIP antiserum (64-720-1), ICN Biochemicals] who acquired it from the same supplier, we set out to perform similar experiments. In our experiments, VIP antiserum at a dilution of 1:25 promptly decreased spontaneous LES tone by 89.9 ± 2.9% and abolished LES relaxation produced by nerve stimulation (1-20 Hz). To determine if this loss of responsiveness was due to a loss of tone, 1 µM carbachol was added to the tissue bath. This restored the LES tone to or slightly above the resting tone before the addition of carbachol, but it did not restore the EFS-induced relaxation. To be sure that the loss of the relaxation in this situation was not simply due to a loss in the responsiveness of the muscle to inhibitory agents, 20 µl of NO · were added to the tissue bath during the control period and after the tissues had been exposed to VIP antiserum and carbachol. During the control period, 20 µl of NO · produced a 75.5 ± 11.3% decrease in tone, but after the tone was restored with carbachol the NO ·-induced relaxation decreased to 24.6 ± 9.1% (n = 4, P < 0.05).

As these experiments were being performed, we noted that the manufacturer formulated this VIP antiserum with 0.1% sodium azide as a preservative. To determine if the azide contaminating the antiserum was responsible for the observed change in LES muscle function, we did a series of experiments in which we added 0.1% sodium azide to the tissue bath to produce a final dilution of 1:25. LES tone dropped, and the nerve-induced LES relaxation was lost, just as was the case with the VIP antiserum formulated with azide.

In an attempt to explore the effects of the VIP antiserum without azide, we tried to separate the compounds by dialyzing 1 ml of VIP antiserum against 3 × 4,000 ml of PBS at 4°C by using Spectra/por molecular porous membrane (132655) (molecular weight cutoff of 6,000-8,000). VIP antiserum treated in this way and used in a final dilution of 1:25 did not decrease LES tone, but it did not antagonize the LES relaxation produced by EFS or exogenous VIP. Because azide alone appeared to reproduce the effects of VIP antiserum containing azide and because we were unable to demonstrate inhibition of VIP- induced or EFS-induced LES relaxation after the azide was dialyzed from the antiserum, we could not use the VIP antiserum to test the hypothesis that VIP is a neurotransmitter controlling relaxation of the opossum LES.

Normal rabbit serum without azide at a final dilution of 1:25 caused a 17.1 ± 5.2% decrease in LES tone, suggesting that some constituent of serum affects LES tone generation.

Effect of calcitonin gene-related peptide antagonist on nerve-induced relaxation of the LES. In a series of earlier studies, we used the calcitonin gene-related peptide (CGRP) receptor antagonist CGRP-(8---37) to determine if CGRP participates in generating the nerve-induced relaxation of the opossum LES (35). In those studies, CGRP-(8---37) did not antagonize either the LES relaxation produced by prolonged (2 min) EFS or the fast component of LES relaxation produced by short trains of EFS. Its effect on the slower component of the LES relaxation was not explored. In the experiments we report here, 1 µM CGRP-(8---37) had no discernable effect on either the fast or the slow component of the LES relaxation (Fig. 5).


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Fig. 5.   Effect of the calcitonin gene-related peptide (CGRP) antagonist CGRP-(8---37) on the biphasic relaxation of the LES. A: graphic demonstration of the frequency-response relationship for the initial, rapid LES relaxation that occurs during EFS. , Control responses. , Responses after tissue was exposed to 1 µM CGRP-(8---37). Error bars indicate SE (n = 5). B: frequency-response relationship for the second, more sustained relaxation that persists after EFS. , Control responses. , Responses after tissue was exposed to 1 µM CGRP-(8---37). Error bars indicate SE (n = 5). CGRP-(8---37) produced no significant change in the responses.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Most if not all investigators studying the mechanisms of LES relaxation focused their attention on the relaxation that occurs during stimulation of intrinsic esophageal nerves; in fact, nearly all that we know about the neuromuscular control of the LES comes from these types of studies. In reviewing some of our old recordings of EFS-induced relaxation, we noticed that the relaxation most often appeared biphasic in nature, with an initial relaxation that occurred during the stimulus and a less pronounced phase of relaxation that lasted up to minutes after the stimulus. In other situations, it appeared as though there was relaxation during the stimulation followed by a brief contraction and then a prolonged relaxation of a lesser amplitude. Such biphasic relaxations have been reported elsewhere in the gut and in other smooth muscle organs (11, 21, 28) and appear to result from the simultaneous action on the muscle of more than one neurotransmitter. These reports and our observations led us to speculate as to whether nerve-induced LES relaxation may actually result from the release of two neurotransmitters. From our previous work, we surmised that NO · should be responsible for most of the relaxation during nerve stimulation, since this relaxation is abolished by inhibitors of NOS, and that a peptide, perhaps VIP or CGRP, might be responsible for the delayed recovery. If such a hypothesis could be proven, it would go a long way to reconcile the controversy regarding the roles of VIP and NO · as mediators of nerve-induced relaxation of the LES. Both VIP and NOS are found in the myenteric neurons supplying the LES, and both VIP and NO relax the LES (8, 15, 27). In fact, histochemical techniques colocalize NOS and VIP to many of the same myenteric neurons in the esophagus (8, 27). Conflicting results come from physiological and biochemical studies meant to determine the roles of VIP and NO in the nerve-induced relaxation of gastrointestinal muscle. One body of evidence supports the hypothesis that the VIP and NO signaling systems interact to produce nerve-mediated relaxation of gastrointestinal muscle (6, 16-19, 22, 24, 29). Another body of evidence, including studies done on the LES of the dog and opossum, suggest that NO ·, not VIP, is the mediator of nerve-induced relaxation (2, 5, 13, 14, 20, 33, 34), and one recent study indicates that both VIP and NO mediate nerve-induced LES relaxation (27). An older literature supports the hypothesis that VIP is the neural mediator of the LES relaxation (4, 15).

The initial goals of the studies reported here were to ascertain whether nerve-induced relaxation of the opossum LES consists of more than one component and to determine if one or both components result from the actions on NO ·. Our studies suggest that EFS-induced relaxation of the LES is biphasic and that the prominence of each component of the relaxation depends on the frequency of the stimulus. There is a transient relaxation that occurs during the stimulus and becomes prominent at lower stimulus frequencies, and there is a relaxation that lasts well after the end of the stimulus and becomes more prominent at higher frequencies of stimulation. Both components of the relaxation are sensitive to TTX, indicating that they were both the result of nerve activation. In a previous study, Jury et al. (23) also identified two EFS-induced relaxations of the LES, but their observation differed from ours in that the relaxation that had the characteristics of our slower relaxation was not sensitive to TTX. This difference may represent a technical dissimilarity. When they used, like we did, short-duration pulses (0.5 ms), they saw rapid EFS-induced LES relaxations that were TTX sensitive. When Jury et al. (23) used longer-duration pulses (5 ms), they generated a slower component of the relaxation that was TTX insensitive. The slow component of the LES relaxation we generated by increasing the frequency of short-duration pulses was TTX sensitive. The reasons for this discrepancy are yet to be determined.

Inhibiting NOS with L-NNA or scavenging NO · with hemoglobin appears to preferentially antagonize the relaxation that occurs during stimulation. Thus most of the first component of the relaxation appears to be mediated by NO ·. At lower frequencies of stimulation, L-NNA and hemoglobin also appear to attenuate the prolonged component of the relaxation. This suggests either that NO · makes some contribution to the generation of the slower component of the relaxation or that we are unable to distinguish the two responses absolutely at lower frequencies of stimulation.

Next, we wished to explore the role played by VIP in the generation of biphasic LES relaxation. Exogenous VIP produced LES relaxation in a concentration-dependent manner. L-NNA, in a concentration that blocked the first component of the relaxation, and hemoglobin, in a concentration that inhibited the relaxation by >50%, did not inhibit VIP-induced relaxation of LES muscle. These data confirm a similar observation made previously by Jury et al. (23) and argue against the hypothesis that VIP released from esophageal nerves stimulates the production of NO in either the smooth muscle or enteric nerves of the LES. This is somewhat different from some other studies (12) and suggests variations in either the neuromuscular apparatus among species or gastrointestinal organs.

We screened the efficacy of a number of putative VIP antagonists before exploring their effects on nerve-induced LES relaxation. None of this group consistently antagonized the relaxation caused by exogenous VIP. Of the group we screened, [Ac-Tyr1,D-Phe2]growth hormone-releasing factor-(1---29) amide (Peninsula 8076) and VIP-(10---28) (Sigma V5381) proved to be inconsistent antagonists in our hands. Neither of these putative antagonists altered the frequency-response relationship for either phase of LES relaxation. Failure of a number of putative VIP antagonists to block nerve-induced relaxation of the LES has been reported before (27), but in those studies the authors did not determine if the antagonists actually affected VIP-induced LES relaxation. It is unclear why these antagonists do not block the effects of exogenous VIP, especially since radioligand binding studies show that they compete effectively for the 125I-labeled VIP binding site on smooth muscle from the opossum internal anal sphincter (7).

Because all of the putative VIP antagonists we used failed to reliably inhibit either VIP-induced or EFS-induced LES relaxation, we attempted to employ a VIP antiserum used previously (27) as another means to explore the role of VIP as a mediator of LES relaxation. We found, as did those other investigators, that VIP antiserum almost completely abolished LES tone. When we explored further, we discovered that azide was present in the antiserum as a preservative. Exposing the tissue to azide in a concentration equal to that present during experiments with antiserum mimicked the effects of the antiserum. Unfortunately, after the antiserum was dialyzed to remove the azide, we were unable to show that the antiserum inhibited LES relaxation produced either by VIP or EFS. In a few experiments, we exposed the tissues to normal serum as a control. Normal serum consistently decreased LES tone but not as dramatically. These data raise the possibility that at least some previous observations of the effects of VIP antiserum on LES function may have been confused by the presence of azide as a preservative. They also indicate that constituents of normal serum may alter LES muscle function. These observations should serve to alert investigators to the hidden pitfalls of using agents like these.

In a previous series of experiments, we used the CGRP antagonist CGRP-(8---37) to explore the role played by CGRP in LES relaxation (35). We found that CGRP did not appear to be involved in the LES relaxation during short- or long-duration EFS. The studies presented here do not support the hypothesis that CGRP participates in the genesis of either component of EFS-induced LES relaxation.

The data presented here suggest that EFS-induced LES relaxation of the opossum esophagus is mediated by more than one neurotransmitter. NO · appears to be the trigger that causes LES relaxation during EFS, and some other agent mediates a prolonged relaxation after the stimulus. Our data do not support the hypothesis that CGRP participates in either phase of relaxation. Whether VIP or another inhibitory agent is involved remains to be determined.


    ACKNOWLEDGEMENTS

This work was supported by a Veterans Affairs Career Development Award to J. A. Murray and a Veterans Affairs Merit Award to J. L. Conklin.


    FOOTNOTES

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

Address for reprint requests and other correspondence: J. L. Conklin, Dept. of Internal Medicine, 4547 John Colloton Pavilion, Univ. of Iowa Hospitals and Clinics, Iowa City, IA 52242. (E-mail: jeffrey-conklin{at}uiowa.edu).

Received 27 August 1998; accepted in final form 25 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Gastroint Liver Physiol 277(3):G548-G554
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